Structured Review

Carl Zeiss gfp expression
Male ssx knockout flies aberrantly express full-length <t>Sxl</t> protein. ( A ) Schematic representation of the reporter gene strategy employed to monitor full-length Sxl protein expression in flies. A T2A-Gal4 encoding sequence was fused to the 3′ end of the Sxl open reading frame (top) to allow expression of a Sxl-T2A-Gal4 fusion protein upon productive splicing (female splicing pattern indicated by dashed lines). Auto-proteolytic cleavage of the T2A sequence in the fusion protein releases the C-terminal Gal4 moiety which activates expression of a <t>GFP</t> reporter gene. ( B–D ) GFP expression in Sxl-T2A-Gal4, UAS-GFP (panel B) and Sxl-T2A-Gal4, UAS-Stinger flies (panels C and D). Control flies with an intact ssx locus are shown on the left, ssx Δ flies in the panels on the right. Arrowheads indicate GFP-positive cells or clonal cell populations in the tibia of the metathoracic leg (panel B) and the 3 rd posterior cell of the wing (panel C) or the midgut (panel D; top: ventral view of the abdomen with the GFP signal visible through the cuticula, bottom: hindgut after manual dissection of the animal). Arrows in panel C indicate GFP-positive nerve projections and neural cell bodies in the L1 and L3 veins of the wing. Scale bars: 0.5mm.
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Images

1) Product Images from "Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing"

Article Title: Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky1284

Male ssx knockout flies aberrantly express full-length Sxl protein. ( A ) Schematic representation of the reporter gene strategy employed to monitor full-length Sxl protein expression in flies. A T2A-Gal4 encoding sequence was fused to the 3′ end of the Sxl open reading frame (top) to allow expression of a Sxl-T2A-Gal4 fusion protein upon productive splicing (female splicing pattern indicated by dashed lines). Auto-proteolytic cleavage of the T2A sequence in the fusion protein releases the C-terminal Gal4 moiety which activates expression of a GFP reporter gene. ( B–D ) GFP expression in Sxl-T2A-Gal4, UAS-GFP (panel B) and Sxl-T2A-Gal4, UAS-Stinger flies (panels C and D). Control flies with an intact ssx locus are shown on the left, ssx Δ flies in the panels on the right. Arrowheads indicate GFP-positive cells or clonal cell populations in the tibia of the metathoracic leg (panel B) and the 3 rd posterior cell of the wing (panel C) or the midgut (panel D; top: ventral view of the abdomen with the GFP signal visible through the cuticula, bottom: hindgut after manual dissection of the animal). Arrows in panel C indicate GFP-positive nerve projections and neural cell bodies in the L1 and L3 veins of the wing. Scale bars: 0.5mm.
Figure Legend Snippet: Male ssx knockout flies aberrantly express full-length Sxl protein. ( A ) Schematic representation of the reporter gene strategy employed to monitor full-length Sxl protein expression in flies. A T2A-Gal4 encoding sequence was fused to the 3′ end of the Sxl open reading frame (top) to allow expression of a Sxl-T2A-Gal4 fusion protein upon productive splicing (female splicing pattern indicated by dashed lines). Auto-proteolytic cleavage of the T2A sequence in the fusion protein releases the C-terminal Gal4 moiety which activates expression of a GFP reporter gene. ( B–D ) GFP expression in Sxl-T2A-Gal4, UAS-GFP (panel B) and Sxl-T2A-Gal4, UAS-Stinger flies (panels C and D). Control flies with an intact ssx locus are shown on the left, ssx Δ flies in the panels on the right. Arrowheads indicate GFP-positive cells or clonal cell populations in the tibia of the metathoracic leg (panel B) and the 3 rd posterior cell of the wing (panel C) or the midgut (panel D; top: ventral view of the abdomen with the GFP signal visible through the cuticula, bottom: hindgut after manual dissection of the animal). Arrows in panel C indicate GFP-positive nerve projections and neural cell bodies in the L1 and L3 veins of the wing. Scale bars: 0.5mm.

Techniques Used: Knock-Out, Expressing, Sequencing, Dissection

Ssx inhibits Sxl-mediated alternative splicing in cultured cells. ( A ) Overexpression of Ssx and GFP in cultured, female Drosophila cells (Kc167). Expression of the transfected constructs (FLAG-3xHA-Ssx and -GFP, as indicated above the lanes) is assessed by Western Blotting using anti-HA (lower panel) or a polyclonal anti-Sxl antibody that cross-reacts with Ssx (top panel). ( B ) RT-PCR analysis of alternative splicing of endogenous Sxl transcripts in transfected Kc167 cells. Splicing products that either lack or contain exon L3 are indicated on the left, molecular weight marker sizes on the right. A control reaction was performed in the absence of reverse transcriptase (panel labelled –RT control) is shown at the bottom. ( C ) Expression of FLAG-3xHA-tagged Sxl, Ssx and GFP in cultured, male Drosophila cells (SL2). Expression levels of the transfected proteins (as indicated above each lane) are analysed by Western Blotting against the HA tag. Sizes of the individual proteins are indicated on the left. ( D ) RT-PCR analysis of alternative splicing of endogenous Sxl transcripts in transfected, male SL2 cells. Transfected constructs are indicated above each lane, non-transfected cells were used as a reference (lane ctrl). Labelling as in panel B.
Figure Legend Snippet: Ssx inhibits Sxl-mediated alternative splicing in cultured cells. ( A ) Overexpression of Ssx and GFP in cultured, female Drosophila cells (Kc167). Expression of the transfected constructs (FLAG-3xHA-Ssx and -GFP, as indicated above the lanes) is assessed by Western Blotting using anti-HA (lower panel) or a polyclonal anti-Sxl antibody that cross-reacts with Ssx (top panel). ( B ) RT-PCR analysis of alternative splicing of endogenous Sxl transcripts in transfected Kc167 cells. Splicing products that either lack or contain exon L3 are indicated on the left, molecular weight marker sizes on the right. A control reaction was performed in the absence of reverse transcriptase (panel labelled –RT control) is shown at the bottom. ( C ) Expression of FLAG-3xHA-tagged Sxl, Ssx and GFP in cultured, male Drosophila cells (SL2). Expression levels of the transfected proteins (as indicated above each lane) are analysed by Western Blotting against the HA tag. Sizes of the individual proteins are indicated on the left. ( D ) RT-PCR analysis of alternative splicing of endogenous Sxl transcripts in transfected, male SL2 cells. Transfected constructs are indicated above each lane, non-transfected cells were used as a reference (lane ctrl). Labelling as in panel B.

Techniques Used: Cell Culture, Over Expression, Expressing, Transfection, Construct, Western Blot, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

2) Product Images from "Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling"

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling

Journal: bioRxiv

doi: 10.1101/826735

Spatiotemporal coordination of hgfa and met expression controls vagus motor axon target selection, see also Supplementary Figure 2 . (A-C) Double hgfa (purple) and tcf21 (brown) RNA in situ hybridization time series showing A-P sequential expression in the PAs. Arrowheads indicate hgfa expression in PAs. Numbers mark PAs. (D-F) met RNA in situ hybridization time series showing A-P expansion over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (G) Quantification of met expression domains over time represented in (D-F). Data represent mean ± SEM. t-test ****P
Figure Legend Snippet: Spatiotemporal coordination of hgfa and met expression controls vagus motor axon target selection, see also Supplementary Figure 2 . (A-C) Double hgfa (purple) and tcf21 (brown) RNA in situ hybridization time series showing A-P sequential expression in the PAs. Arrowheads indicate hgfa expression in PAs. Numbers mark PAs. (D-F) met RNA in situ hybridization time series showing A-P expansion over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (G) Quantification of met expression domains over time represented in (D-F). Data represent mean ± SEM. t-test ****P

Techniques Used: Expressing, Selection, RNA In Situ Hybridization

(A-C) hoxb5a RNA in situ hybridization time series showing hoxb5a expression receding towards the posterior over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (D) Quantification of hoxb5a expression domains over time represented in (A-C). Data represent mean ± SEM. t-test ****P
Figure Legend Snippet: (A-C) hoxb5a RNA in situ hybridization time series showing hoxb5a expression receding towards the posterior over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (D) Quantification of hoxb5a expression domains over time represented in (A-C). Data represent mean ± SEM. t-test ****P

Techniques Used: RNA In Situ Hybridization, Expressing

Retinoic Acid is a putative regulator of A-P vagus motor neuron identity (A-B) Differential gene expression between anterior and posterior mX neurons. (A) Representative anterior (A) and posterior (A’) photoconverted (magenta) regions collected for RNAseq analysis. (B) Volcano plot of RNAseq data indicating mRNAs enriched in anterior (left) or posterior (right) mX neurons. Dashed lines indicate significance threshold for a false discovery rate of 10% (y-axis) and a fold change of 1.5 (x-axis). Blue and red dots represent significantly differentially expressed genes. Red dots represent genes indicative of differential Retinoic Acid signaling between populations. (C-E) RNA In situ hybridization of selected genes identified in (B). In each image, mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. hoxb5a (C) and dhrs3b (D) mRNAs are enriched in posterior mX neurons. crabp1b (E) mRNA is enriched in anterior mX neurons. (F) The retinoic acid-responsive RARE:GFP transgene (green) is expressed in posterior, but not anterior, mX neurons (magenta). All images are lateral views oriented with anterior to left. Scale bars = 50 μm.
Figure Legend Snippet: Retinoic Acid is a putative regulator of A-P vagus motor neuron identity (A-B) Differential gene expression between anterior and posterior mX neurons. (A) Representative anterior (A) and posterior (A’) photoconverted (magenta) regions collected for RNAseq analysis. (B) Volcano plot of RNAseq data indicating mRNAs enriched in anterior (left) or posterior (right) mX neurons. Dashed lines indicate significance threshold for a false discovery rate of 10% (y-axis) and a fold change of 1.5 (x-axis). Blue and red dots represent significantly differentially expressed genes. Red dots represent genes indicative of differential Retinoic Acid signaling between populations. (C-E) RNA In situ hybridization of selected genes identified in (B). In each image, mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. hoxb5a (C) and dhrs3b (D) mRNAs are enriched in posterior mX neurons. crabp1b (E) mRNA is enriched in anterior mX neurons. (F) The retinoic acid-responsive RARE:GFP transgene (green) is expressed in posterior, but not anterior, mX neurons (magenta). All images are lateral views oriented with anterior to left. Scale bars = 50 μm.

Techniques Used: Expressing, RNA In Situ Hybridization

3) Product Images from "Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection"

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection

Journal:

doi: 10.1523/JNEUROSCI.3200-07.2008

p21 is sufficient for protection of neurons from oxidative stress-induced death. A , Western blot analysis to detect relative levels of GFP or p21-GFP fusion protein in lysates from HT22 murine hippocampal cells stably transfected with either pEGFP or
Figure Legend Snippet: p21 is sufficient for protection of neurons from oxidative stress-induced death. A , Western blot analysis to detect relative levels of GFP or p21-GFP fusion protein in lysates from HT22 murine hippocampal cells stably transfected with either pEGFP or

Techniques Used: Western Blot, Stable Transfection, Transfection

4) Product Images from "Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes"

Article Title: Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes

Journal: bioRxiv

doi: 10.1101/2020.07.20.212050

CSR-1a silences a repetitive somatic transgene. A) Schematic of the forward genetic screen used to uncover genes that repress rpn-2p::GFP expression. B) Viable alleles recovered from the forward genetic screen. C) Schematic representation of the csr-1a alleles recovered from the forward genetic screen. D) Fluorescence micrographs of the non-mutagenized reporter strain and the reporter after introduction of csr-1a [G120*] . Scale bar, 100μm. E) Box plot of normalized median relative fluorescence of the reporter strain and the reporter with csr-1a [G120*] strain (with Tukey whiskers). **** indicates significance of p
Figure Legend Snippet: CSR-1a silences a repetitive somatic transgene. A) Schematic of the forward genetic screen used to uncover genes that repress rpn-2p::GFP expression. B) Viable alleles recovered from the forward genetic screen. C) Schematic representation of the csr-1a alleles recovered from the forward genetic screen. D) Fluorescence micrographs of the non-mutagenized reporter strain and the reporter after introduction of csr-1a [G120*] . Scale bar, 100μm. E) Box plot of normalized median relative fluorescence of the reporter strain and the reporter with csr-1a [G120*] strain (with Tukey whiskers). **** indicates significance of p

Techniques Used: Expressing, Fluorescence

5) Product Images from "Combinatorial Action of Temporally Segregated Transcription Factors"

Article Title: Combinatorial Action of Temporally Segregated Transcription Factors

Journal: Developmental Cell

doi: 10.1016/j.devcel.2020.09.002

TBX-37/38 Establish a Differentially Accessible State of the lsy-6 Locus (A) Schematic of the embryo-labeling strategy for ABa and ABp isolation. The indicated time points (90, 200, and 350 min) were used for the experiments in (B) and (C). Representative image of an embryo at the 8 ABa stage carrying the three-reporter combination ( Figures S3 A–S3C). (B) (Top) Aggregated GFP-TBX-37/38 ChIP-seq signal over the lsy-6 locus and flanking sequences. (Middle) ATAC-seq signal in ABa- and ABp-derived cells at three different time points showing ABa-specific accessibility of lsy-6 , overlapping with the TBX-37/38 binding site (red shading); two biological replicates were analyzed per condition. For reference, downstream locus shows equal accessibility in both lineages (also Figure S3 D). Number of ABa/ABp descendants at the different time points are shown. (Bottom) ATAC-seq signal from sorted ASEL and ASER shows accessibility in ASEL upstream of lsy-6 , overlapping with a CHE-1 binding site (blue shading). Peaks called by MACS2 are marked with a bar. (C) Close-up view of the lsy-6 locus and its ATAC-seq signal in ABa, ABp, ASEL, and ASER isolated from wild-type or Δtbs embryos (deletion marked with a red line); two biological replicates were analyzed per condition. Loss of TBX-37/38 binding sites causes loss of accessibility of the CHE-1 binding site in mature ASEs. For Δtbs embryos, ASEL and ASER cannot be distinguished as lsy-6 is not expressed and the cells become symmetric ( Figure 2 B); the ASE ATAC-seq in this case was done on che-1 prom ::mCherry -expressing cells, and the same track is shown in duplicate under ASEL and ASER. (D) Correlation analysis between ATAC-seq data from wild-type or Δtbs embryos shows that datasets are highly similar and differ almost exclusively in their signal over the lsy-6 locus. Plotted are signals in log 2 (cpm) for all peaks called by MACS2 in at least one ATAC-seq sample. The p values for the called lsy-6 peaks in ABa (overlapping with TBX-binding sites, red) and ASEL (overlapping with CHE-1 binding site, blue) are shown.
Figure Legend Snippet: TBX-37/38 Establish a Differentially Accessible State of the lsy-6 Locus (A) Schematic of the embryo-labeling strategy for ABa and ABp isolation. The indicated time points (90, 200, and 350 min) were used for the experiments in (B) and (C). Representative image of an embryo at the 8 ABa stage carrying the three-reporter combination ( Figures S3 A–S3C). (B) (Top) Aggregated GFP-TBX-37/38 ChIP-seq signal over the lsy-6 locus and flanking sequences. (Middle) ATAC-seq signal in ABa- and ABp-derived cells at three different time points showing ABa-specific accessibility of lsy-6 , overlapping with the TBX-37/38 binding site (red shading); two biological replicates were analyzed per condition. For reference, downstream locus shows equal accessibility in both lineages (also Figure S3 D). Number of ABa/ABp descendants at the different time points are shown. (Bottom) ATAC-seq signal from sorted ASEL and ASER shows accessibility in ASEL upstream of lsy-6 , overlapping with a CHE-1 binding site (blue shading). Peaks called by MACS2 are marked with a bar. (C) Close-up view of the lsy-6 locus and its ATAC-seq signal in ABa, ABp, ASEL, and ASER isolated from wild-type or Δtbs embryos (deletion marked with a red line); two biological replicates were analyzed per condition. Loss of TBX-37/38 binding sites causes loss of accessibility of the CHE-1 binding site in mature ASEs. For Δtbs embryos, ASEL and ASER cannot be distinguished as lsy-6 is not expressed and the cells become symmetric ( Figure 2 B); the ASE ATAC-seq in this case was done on che-1 prom ::mCherry -expressing cells, and the same track is shown in duplicate under ASEL and ASER. (D) Correlation analysis between ATAC-seq data from wild-type or Δtbs embryos shows that datasets are highly similar and differ almost exclusively in their signal over the lsy-6 locus. Plotted are signals in log 2 (cpm) for all peaks called by MACS2 in at least one ATAC-seq sample. The p values for the called lsy-6 peaks in ABa (overlapping with TBX-binding sites, red) and ASEL (overlapping with CHE-1 binding site, blue) are shown.

Techniques Used: Labeling, Isolation, Chromatin Immunoprecipitation, Derivative Assay, Binding Assay, Expressing

Transcription over the lsy-6 Locus Occurs Bidirectionally and Is Required for Priming (A) smFISH on embryos carrying the lsy-6::yfp fosmid showed robust antisense transcription of the lsy-6 locus (see Figure S4 for sense transcript and dependence on TBX-37/38 and their binding sites). Representative images are shown (n ≥ 10). Dashed boxes indicate zoomed regions showing bright nuclear foci. At the bean stage, the mature ASEL neuron is outlined as determined by strong signal against the sense yfp transcript. Scale bars represent 10 μm. (B) The TBX-37/38 binding sites were replaced by five UAS sites in the context of the lsy-6::gfp fosmid reporter. YFP expression was restored exclusively in ASEL by expression of the GAL4 DBD -VP64 transcriptional activator under the tbx-37 prom ( Table S2 ). Four independent, extrachromosomal transgenic lines were scored for each condition. Number of animals scored per line are shown. (C) Five UAS sites were inserted downstream of the TBX-37/38 binding sites in the context of the lsy-6::gfp fosmid reporter. Expression of GAL4 DBD -UNC-37 under the tbx-37 prom prevented later expression in ASEL ( Table S2 ). Two independent, extrachromosomal transgenic lines were scored. (D) Embryos carrying the integrated lsy-6::gfp::Δtbs::5xUAS reporter and a transgene containing GAL4 DBD -VP64 under a heat-shock promoter were subjected to heat shock at the indicated times (dashed lines). GAL4 DBD -VP64 expression in the whole embryo at early time points induced lsy-6::gfp::Δtbs::5xUAS expression in both ASEs. Beyond the 180-min time point, GAL4 DBD -VP64 progressively lost ability to activate lsy-6::gfp::Δtbs::5xUAS expression. Plot shows proportion and SEP (n ≥ 6 per time point). Representative images are shown for the 90- and 300-min time points. Arrowheads point to sporadic ectopic expression in two cells in the tail upon heat-shock treatment (independently of GAL4-VP64 expression). Scale bars represent 20 μm.
Figure Legend Snippet: Transcription over the lsy-6 Locus Occurs Bidirectionally and Is Required for Priming (A) smFISH on embryos carrying the lsy-6::yfp fosmid showed robust antisense transcription of the lsy-6 locus (see Figure S4 for sense transcript and dependence on TBX-37/38 and their binding sites). Representative images are shown (n ≥ 10). Dashed boxes indicate zoomed regions showing bright nuclear foci. At the bean stage, the mature ASEL neuron is outlined as determined by strong signal against the sense yfp transcript. Scale bars represent 10 μm. (B) The TBX-37/38 binding sites were replaced by five UAS sites in the context of the lsy-6::gfp fosmid reporter. YFP expression was restored exclusively in ASEL by expression of the GAL4 DBD -VP64 transcriptional activator under the tbx-37 prom ( Table S2 ). Four independent, extrachromosomal transgenic lines were scored for each condition. Number of animals scored per line are shown. (C) Five UAS sites were inserted downstream of the TBX-37/38 binding sites in the context of the lsy-6::gfp fosmid reporter. Expression of GAL4 DBD -UNC-37 under the tbx-37 prom prevented later expression in ASEL ( Table S2 ). Two independent, extrachromosomal transgenic lines were scored. (D) Embryos carrying the integrated lsy-6::gfp::Δtbs::5xUAS reporter and a transgene containing GAL4 DBD -VP64 under a heat-shock promoter were subjected to heat shock at the indicated times (dashed lines). GAL4 DBD -VP64 expression in the whole embryo at early time points induced lsy-6::gfp::Δtbs::5xUAS expression in both ASEs. Beyond the 180-min time point, GAL4 DBD -VP64 progressively lost ability to activate lsy-6::gfp::Δtbs::5xUAS expression. Plot shows proportion and SEP (n ≥ 6 per time point). Representative images are shown for the 90- and 300-min time points. Arrowheads point to sporadic ectopic expression in two cells in the tail upon heat-shock treatment (independently of GAL4-VP64 expression). Scale bars represent 20 μm.

Techniques Used: Binding Assay, Expressing, Transgenic Assay

6) Product Images from "Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper"

Article Title: Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper

Journal: Plant Biotechnology Reports

doi: 10.1007/s11816-011-0168-1

Western blot analysis and RT-PCR products from GFP-expressing transgenic and non-transgenic peppers. a M pre-stained marker, lane 1 2211 (non-GM), lane 2 2213 (GM), lane 3 2214 (GM), lane 4 2216 (non-GM), lane 5 2219 (GM), P positive control (GFP purified), N negative (coat protein). Arrow indicates a protein band of about 30 kDa. b GFP expression in T 1 whole peppers versus non-transgenic peppers. c RT-PCR of GFP gene from transgenic pepper plants. Arrow indicates the amplified DNA band of GFP gene (573 bp). Lanes 1 and 4 non-GM, lanes 2, 3, 5 GM
Figure Legend Snippet: Western blot analysis and RT-PCR products from GFP-expressing transgenic and non-transgenic peppers. a M pre-stained marker, lane 1 2211 (non-GM), lane 2 2213 (GM), lane 3 2214 (GM), lane 4 2216 (non-GM), lane 5 2219 (GM), P positive control (GFP purified), N negative (coat protein). Arrow indicates a protein band of about 30 kDa. b GFP expression in T 1 whole peppers versus non-transgenic peppers. c RT-PCR of GFP gene from transgenic pepper plants. Arrow indicates the amplified DNA band of GFP gene (573 bp). Lanes 1 and 4 non-GM, lanes 2, 3, 5 GM

Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Staining, Marker, Positive Control, Purification, Amplification

GFP expression in T 1 fruits and T 2 seeds obtained by self-crossing of T 1 peppers. Pictures were taken using LAS-3000 luminescent image analyzer. N non-GM pepper, T transgenic pepper
Figure Legend Snippet: GFP expression in T 1 fruits and T 2 seeds obtained by self-crossing of T 1 peppers. Pictures were taken using LAS-3000 luminescent image analyzer. N non-GM pepper, T transgenic pepper

Techniques Used: Expressing, Transgenic Assay

7) Product Images from "Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages ▿"

Article Title: Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages ▿

Journal:

doi: 10.1128/IAI.00183-08

Apoptosis of BMDMs, as measured by anti-cleaved caspase-3 immunostaining or TUNEL. BMDMs were infected with the indicated strains for 6 h and GFP expression was induced during the last hour as described in the legend for Fig. . Cleaved
Figure Legend Snippet: Apoptosis of BMDMs, as measured by anti-cleaved caspase-3 immunostaining or TUNEL. BMDMs were infected with the indicated strains for 6 h and GFP expression was induced during the last hour as described in the legend for Fig. . Cleaved

Techniques Used: Immunostaining, TUNEL Assay, Infection, Expressing

Survival and replication of Y. pseudotuberculosis inside BMDMs, as determined by GFP induction and viable count assays. BMDMs were infected with the indicated strains for 1, 4, or 24 h, as described for Fig. . (A) Overlaid images of green
Figure Legend Snippet: Survival and replication of Y. pseudotuberculosis inside BMDMs, as determined by GFP induction and viable count assays. BMDMs were infected with the indicated strains for 1, 4, or 24 h, as described for Fig. . (A) Overlaid images of green

Techniques Used: Infection

8) Product Images from "poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance"

Article Title: poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance

Journal: bioRxiv

doi: 10.1101/2019.12.31.891960

pUG tails convert otherwise inert RNAs into agents of gene silencing. a , Fluorescent micrographs showing -1 to -3 oocytes of adult rde-1(ne219); gfp::h2b animals injected in the germline with RNAs consisting of the indicated 3’ terminal repeats appended to the first 369nt of gfp mRNA. % of progeny with gfp silenced was counted. b, oma-1(zu405ts) animals lay arrested embryos at 20°C unless oma-1(zu405ts) is silenced 14 . Adult rde-1(ne219); oma-1(zu405ts) animals were injected with RNAs consisting of the indicated 3’ terminal repeats appended to the first 541nt of oma-1 mRNA. c, Adult rde-1(ne219); oma-1(zu405ts) animals were injected with the same oma-1 mRNA fragment as in b with varying 3’ pUG tail length, different UG repeat sequences or with the pUG sequence appended to the 3’ end, 5’ end or in the middle of the oma-1 mRNA. b-c, 5 progeny per injected animal were pooled and the % hatched embryos (# of hatched embryos/total embryos laid) was counted. Insets show injected RNAs run on a 2% agarose gel to assess RNA integrity. n=5-15 injected animals. a-c, Repeats were 36nt in length unless otherwise indicated. Error bars are standard deviations (s.d.) of the mean.
Figure Legend Snippet: pUG tails convert otherwise inert RNAs into agents of gene silencing. a , Fluorescent micrographs showing -1 to -3 oocytes of adult rde-1(ne219); gfp::h2b animals injected in the germline with RNAs consisting of the indicated 3’ terminal repeats appended to the first 369nt of gfp mRNA. % of progeny with gfp silenced was counted. b, oma-1(zu405ts) animals lay arrested embryos at 20°C unless oma-1(zu405ts) is silenced 14 . Adult rde-1(ne219); oma-1(zu405ts) animals were injected with RNAs consisting of the indicated 3’ terminal repeats appended to the first 541nt of oma-1 mRNA. c, Adult rde-1(ne219); oma-1(zu405ts) animals were injected with the same oma-1 mRNA fragment as in b with varying 3’ pUG tail length, different UG repeat sequences or with the pUG sequence appended to the 3’ end, 5’ end or in the middle of the oma-1 mRNA. b-c, 5 progeny per injected animal were pooled and the % hatched embryos (# of hatched embryos/total embryos laid) was counted. Insets show injected RNAs run on a 2% agarose gel to assess RNA integrity. n=5-15 injected animals. a-c, Repeats were 36nt in length unless otherwise indicated. Error bars are standard deviations (s.d.) of the mean.

Techniques Used: Injection, Sequencing, Agarose Gel Electrophoresis

Endogenous RNAs are pUGylated and localize to germline Mutator foci. a , Total RNA isolated from adult wild-type or rde-3 mutant animals was subjected to Tc1 pUG PCR analysis ( Fig. 1a ). Rescue strategies are described in the Main text and Methods. b, A 36nt pUG tail was appended to a 338nt Tc1 RNA fragment and this Tc1 pUG RNA was injected into germlines of rde-3(-); unc-22::tc1 animals with a co-injection marker. 25 co-injection marker expressing progeny were pooled per injected animal. Each data point represents the # of mobile progeny (indicating Tc1 mobilized from unc-22 ) per pool. Error bars represent s.d. c-e, Fluorescent micrographs of adult pachytene germ cell nuclei. c, Wild-type or rde-3(-) animals expressing a marker of chromatin (mCherry:HIS-58, magenta) and C38D9.2::GFP (green), which is expressed diffusely in the germline syncytium, wherein germ cell nuclei share a common cytoplasm. d, RNA FISH to detect pUG RNAs (pUG RNA FISH) was performed on germlines dissected from wild-type or rde-3(-) animals using an 18nt long poly(AC) oligo conjugated to Alexa 647 (magenta). RNA FISH to detect ama-1 mRNA (green) was performed simultaneously as a positive control. DNA was stained with DAPI (blue). e, pUG RNA FISH (magenta) and immunofluorescence to detect a GFP- and degron-tagged RDE-3 (green). DNA was stained with DAPI (blue). f, Tc1, dpy-11 , and oma-1 pUG PCR was performed on total RNA isolated from glp-1(q224 or ts) animals grown at 15°C (permissive temperature, germ cells present) or 25°C (non-permissive temperature,
Figure Legend Snippet: Endogenous RNAs are pUGylated and localize to germline Mutator foci. a , Total RNA isolated from adult wild-type or rde-3 mutant animals was subjected to Tc1 pUG PCR analysis ( Fig. 1a ). Rescue strategies are described in the Main text and Methods. b, A 36nt pUG tail was appended to a 338nt Tc1 RNA fragment and this Tc1 pUG RNA was injected into germlines of rde-3(-); unc-22::tc1 animals with a co-injection marker. 25 co-injection marker expressing progeny were pooled per injected animal. Each data point represents the # of mobile progeny (indicating Tc1 mobilized from unc-22 ) per pool. Error bars represent s.d. c-e, Fluorescent micrographs of adult pachytene germ cell nuclei. c, Wild-type or rde-3(-) animals expressing a marker of chromatin (mCherry:HIS-58, magenta) and C38D9.2::GFP (green), which is expressed diffusely in the germline syncytium, wherein germ cell nuclei share a common cytoplasm. d, RNA FISH to detect pUG RNAs (pUG RNA FISH) was performed on germlines dissected from wild-type or rde-3(-) animals using an 18nt long poly(AC) oligo conjugated to Alexa 647 (magenta). RNA FISH to detect ama-1 mRNA (green) was performed simultaneously as a positive control. DNA was stained with DAPI (blue). e, pUG RNA FISH (magenta) and immunofluorescence to detect a GFP- and degron-tagged RDE-3 (green). DNA was stained with DAPI (blue). f, Tc1, dpy-11 , and oma-1 pUG PCR was performed on total RNA isolated from glp-1(q224 or ts) animals grown at 15°C (permissive temperature, germ cells present) or 25°C (non-permissive temperature,

Techniques Used: Isolation, Mutagenesis, Polymerase Chain Reaction, Injection, Marker, Expressing, Fluorescence In Situ Hybridization, Positive Control, Staining, Immunofluorescence

pUG RNA-directed gene silencing is specific. a , rde-1(ne219); oma-1(zu405ts) animals were injected with either an oma-1 or gfp pUG RNA. Inset shows injected RNAs run on a 2% agarose gel to assess RNA integrity. b, rde-1(ne219); gfp::h2b animals were injected with either an oma-1 or gfp pUG RNA. % embryonic arrest (a) and % gfp silencing (b) were scored. All pUG tails were 36nt in length. n = 6-10 injected animals.
Figure Legend Snippet: pUG RNA-directed gene silencing is specific. a , rde-1(ne219); oma-1(zu405ts) animals were injected with either an oma-1 or gfp pUG RNA. Inset shows injected RNAs run on a 2% agarose gel to assess RNA integrity. b, rde-1(ne219); gfp::h2b animals were injected with either an oma-1 or gfp pUG RNA. % embryonic arrest (a) and % gfp silencing (b) were scored. All pUG tails were 36nt in length. n = 6-10 injected animals.

Techniques Used: Injection, Agarose Gel Electrophoresis

RNAi-triggered pUGylation is general and sequence-specific. a , gfp::h2b, rde-3(-); gfp::h2b and WT (no gfp::h2b ) animals were fed E.coli expressing either empty vector control or gfp dsRNA. b, WT and rde-3(-) animals were fed E.coli expressing empty vector control and either oma-1 or dpy-11 dsRNA. gfp ( a ), dpy-11 and oma-1 ( b ) pUG RNAs were detected using the assay outlined in Fig. 1a .
Figure Legend Snippet: RNAi-triggered pUGylation is general and sequence-specific. a , gfp::h2b, rde-3(-); gfp::h2b and WT (no gfp::h2b ) animals were fed E.coli expressing either empty vector control or gfp dsRNA. b, WT and rde-3(-) animals were fed E.coli expressing empty vector control and either oma-1 or dpy-11 dsRNA. gfp ( a ), dpy-11 and oma-1 ( b ) pUG RNAs were detected using the assay outlined in Fig. 1a .

Techniques Used: Sequencing, Expressing, Plasmid Preparation

pUG RNAs and siRNAs cooperate to drive heritable gene silencing. a , oma-1 pUG PCR was performed on RNA isolated from four generations of descendants (F 1 -F 4 ) derived from oma-1 dsRNA-treated animals. b, rde-1(ne219); gfp::h2b animals were injected with a gfp pUG RNA and gfp expression was monitored for six generations. c, MAGO12 animals, which harbor deletions in all twelve wago genes, were treated with oma-1 dsRNA. oma-1 pUG PCR was performed on total RNA from dsRNA-treated animals (P 0 ) and their progeny (F 1 ). Note: pUG RNAs appear longer in MAGO12 animals (see Extended Data Fig. 9 legend). d, c38d9.2 and Tc1 pUG RNA expression levels were quantified in embryos harvested from wild-type, MAGO12, or rde-3(-) animals. Shown is the fold change normalized to rde-3(-). e, rde-1(ne219); oma-1(zu405ts) animals were injected with an oma-1(SNP) pUG RNA. pUG RNAs were Sanger sequenced from F 2 progeny to determine the presence or absence of the SNP. f, Wild-type and rde-3(ne298) animals subjected to oma-1 RNAi were crossed and F 2 progeny were genotyped (not shown). RNA isolated from populations of rde-3(+) or rde-3(ne298) F 3 animals (3 biological replicates) was subjected to oma-1 pUG PCR. g, Model. Two major phases of the pUG RNA pathway, initiation and maintenance, are shown. Initiation : exogenous and constitutive (i.e. genomically-encoded such as dsRNA, piRNAs) triggers direct RDE-3 to pUGylate RNAs previously fragmented by RNAi, and possibly other, systems. Maintenance : pUG RNA are templates for RdRPs to make 2° siRNAs. Argonaute proteins (termed WAGOs) bind these 2° siRNAs and: 1) target homologous RNAs for transcriptional and translational silencing (previous work 25 , 30 , 38 , 39 ), and 2) direct the cleavage and de novo RDE-3-mediated pUGylation of additional mRNAs (this work). In this way, cycles of pUG RNA-based siRNA production and siRNA-directed mRNA pUGylation form a silencing loop, which is maintained over time and across generations to mediate stable gene silencing. pUG/siRNA cycling likely occurs in germline perinuclear condensates called Mutator foci.
Figure Legend Snippet: pUG RNAs and siRNAs cooperate to drive heritable gene silencing. a , oma-1 pUG PCR was performed on RNA isolated from four generations of descendants (F 1 -F 4 ) derived from oma-1 dsRNA-treated animals. b, rde-1(ne219); gfp::h2b animals were injected with a gfp pUG RNA and gfp expression was monitored for six generations. c, MAGO12 animals, which harbor deletions in all twelve wago genes, were treated with oma-1 dsRNA. oma-1 pUG PCR was performed on total RNA from dsRNA-treated animals (P 0 ) and their progeny (F 1 ). Note: pUG RNAs appear longer in MAGO12 animals (see Extended Data Fig. 9 legend). d, c38d9.2 and Tc1 pUG RNA expression levels were quantified in embryos harvested from wild-type, MAGO12, or rde-3(-) animals. Shown is the fold change normalized to rde-3(-). e, rde-1(ne219); oma-1(zu405ts) animals were injected with an oma-1(SNP) pUG RNA. pUG RNAs were Sanger sequenced from F 2 progeny to determine the presence or absence of the SNP. f, Wild-type and rde-3(ne298) animals subjected to oma-1 RNAi were crossed and F 2 progeny were genotyped (not shown). RNA isolated from populations of rde-3(+) or rde-3(ne298) F 3 animals (3 biological replicates) was subjected to oma-1 pUG PCR. g, Model. Two major phases of the pUG RNA pathway, initiation and maintenance, are shown. Initiation : exogenous and constitutive (i.e. genomically-encoded such as dsRNA, piRNAs) triggers direct RDE-3 to pUGylate RNAs previously fragmented by RNAi, and possibly other, systems. Maintenance : pUG RNA are templates for RdRPs to make 2° siRNAs. Argonaute proteins (termed WAGOs) bind these 2° siRNAs and: 1) target homologous RNAs for transcriptional and translational silencing (previous work 25 , 30 , 38 , 39 ), and 2) direct the cleavage and de novo RDE-3-mediated pUGylation of additional mRNAs (this work). In this way, cycles of pUG RNA-based siRNA production and siRNA-directed mRNA pUGylation form a silencing loop, which is maintained over time and across generations to mediate stable gene silencing. pUG/siRNA cycling likely occurs in germline perinuclear condensates called Mutator foci.

Techniques Used: Polymerase Chain Reaction, Isolation, Derivative Assay, Injection, Expressing, RNA Expression

9) Product Images from "Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti"

Article Title: Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Journal: Scientific Reports

doi: 10.1038/s41598-020-72489-3

Stable transfection of the B. microti parasite. ( A ) Pictorial representation of a double homologous recombination strategy used for creating a recombinant B. microti parasite expressing GFP and mCherry gene in a same parasite. ( B ) Diagnostic PCR. The primer P21 and P22 is specific for the 5′ integration event and it should not give any PCR product on WT gDNA. The primer P23 and P24 is specific for the 3′ integration event. A PCR product of right size in the diagnostic PCR confirmed the integration of GFP-mCherry cassette at the right locus in B. microti parasite. Image shown is cropped and pixel inverted for better visualization and printing. Original image is provided in the supplementary file for clarity and comparison. ( C ) Southern blot analysis of transfected parasites. The DIG labeled GFP probe was used to detect the integration event in the digested gDNA. A band of ~ 10.5 kb was detected in the transgenic B. microti parasite. Wild-type parasites do not have the gene for GFP hence no bands are expected. Image shown is cropped and color converted to grey scale for better visualization and printing. Original image (color) is provided in the supplementary file for clarity and comparison. ( D ) Confirmation of a stable transfection in B. microti by fluorescence microscopy of the parasites expressing the GFP and mCherry gene. Green and red images correspond to the parasite expressing GFP and mCherry gene within a stably transfected parasite. Merged represents the overlap of all the images. Scale bar represents 5 μm.
Figure Legend Snippet: Stable transfection of the B. microti parasite. ( A ) Pictorial representation of a double homologous recombination strategy used for creating a recombinant B. microti parasite expressing GFP and mCherry gene in a same parasite. ( B ) Diagnostic PCR. The primer P21 and P22 is specific for the 5′ integration event and it should not give any PCR product on WT gDNA. The primer P23 and P24 is specific for the 3′ integration event. A PCR product of right size in the diagnostic PCR confirmed the integration of GFP-mCherry cassette at the right locus in B. microti parasite. Image shown is cropped and pixel inverted for better visualization and printing. Original image is provided in the supplementary file for clarity and comparison. ( C ) Southern blot analysis of transfected parasites. The DIG labeled GFP probe was used to detect the integration event in the digested gDNA. A band of ~ 10.5 kb was detected in the transgenic B. microti parasite. Wild-type parasites do not have the gene for GFP hence no bands are expected. Image shown is cropped and color converted to grey scale for better visualization and printing. Original image (color) is provided in the supplementary file for clarity and comparison. ( D ) Confirmation of a stable transfection in B. microti by fluorescence microscopy of the parasites expressing the GFP and mCherry gene. Green and red images correspond to the parasite expressing GFP and mCherry gene within a stably transfected parasite. Merged represents the overlap of all the images. Scale bar represents 5 μm.

Techniques Used: Stable Transfection, Homologous Recombination, Recombinant, Expressing, Diagnostic Assay, Polymerase Chain Reaction, Southern Blot, Transfection, Labeling, Transgenic Assay, Fluorescence, Microscopy

Transient transfection of B. microti parasite. ( A ) Genomic analysis of a BM-CTQ41297 promoter in the parasite. ( B ) Pictorial representation of a construct map for control plasmid. ( C ) Images represent the B. microti parasites transfected with the control plasmid. DAPI staining corresponds to the nucleus of the parasite within RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. (D) Map of a transient transfection construct containing GFP gene. ( E ) Green flourescence corresponds to the transfected parasite expressing GFP, DAPI staining represents the nucleus of the parasite, and DIC image showing a parasitized RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. ( F ) Map of a mCherry construct used for parasite transient transfection. ( G ) On the right side red flourescence corresponds to the transfected parasite expressing mCherry. DAPI staining (Blue) corresponds to the nucleus of the parasite, and DIC is showing a parasitized RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. ( H ) Construct map of a plasmid containing GFP and mCherry gene on either side of a BM-CTQ41297 promoter. ( I ) Green and red fluorescence signal correspond to the same parasite expressing GFP and mCherry. DIC image represents the parasitized RBC and merged is the overlap of all the images. Scale bar represents 5 μm.
Figure Legend Snippet: Transient transfection of B. microti parasite. ( A ) Genomic analysis of a BM-CTQ41297 promoter in the parasite. ( B ) Pictorial representation of a construct map for control plasmid. ( C ) Images represent the B. microti parasites transfected with the control plasmid. DAPI staining corresponds to the nucleus of the parasite within RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. (D) Map of a transient transfection construct containing GFP gene. ( E ) Green flourescence corresponds to the transfected parasite expressing GFP, DAPI staining represents the nucleus of the parasite, and DIC image showing a parasitized RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. ( F ) Map of a mCherry construct used for parasite transient transfection. ( G ) On the right side red flourescence corresponds to the transfected parasite expressing mCherry. DAPI staining (Blue) corresponds to the nucleus of the parasite, and DIC is showing a parasitized RBC. Merged image represents the overlap of all images. Scale bar represents 5 μm. ( H ) Construct map of a plasmid containing GFP and mCherry gene on either side of a BM-CTQ41297 promoter. ( I ) Green and red fluorescence signal correspond to the same parasite expressing GFP and mCherry. DIC image represents the parasitized RBC and merged is the overlap of all the images. Scale bar represents 5 μm.

Techniques Used: Transfection, Construct, Plasmid Preparation, Staining, Expressing, Fluorescence

Plasmodium berghei DHFR promoter is recognized by the B. microti parasite. ( A ) The map of an empty plasmid construct used as a control. ( B ) The map of a transient transfection construct containing a Pb-DHFR promoter and a GFP gene. ( C ) The green image represents the parasite expressing GFP and DAPI represent the parasite nucleus stained with DAPI. DIC is showing the infected RBC. Control represents the parasite transfected with control plasmid without any DHFR promoter and GFP gene. Scale bar represents 5 μm.
Figure Legend Snippet: Plasmodium berghei DHFR promoter is recognized by the B. microti parasite. ( A ) The map of an empty plasmid construct used as a control. ( B ) The map of a transient transfection construct containing a Pb-DHFR promoter and a GFP gene. ( C ) The green image represents the parasite expressing GFP and DAPI represent the parasite nucleus stained with DAPI. DIC is showing the infected RBC. Control represents the parasite transfected with control plasmid without any DHFR promoter and GFP gene. Scale bar represents 5 μm.

Techniques Used: Plasmid Preparation, Construct, Transfection, Expressing, Staining, Infection

10) Product Images from "The GATA Transcription Factor egl-27 Delays Aging by Promoting Stress Resistance in Caenorhabditis elegans"

Article Title: The GATA Transcription Factor egl-27 Delays Aging by Promoting Stress Resistance in Caenorhabditis elegans

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003108

egl-27 acts downstream of daf-2 /ILR, daf-16 /FOXO, and elt-3 /GATA. (A) representative images of egl-27::mCherry hermaphrodites at day 2 of adulthood showing median expression levels for each group. (B) egl-27 expression is increased in daf-2(e1370) mutants (p = 1.5×10 −11 vs. control) and this increase is suppressed in daf-2(e1370); daf-16(mu86) double mutants (p = 2.9×10 −17 vs. daf-2(e1370) worms). egl-27 expression is reduced in elt-3(vp1) mutants (p = 2.3×10 −8 vs. control) and the addition of daf-2(e1370) in daf-2(e1370); elt-3(vp1) double mutants is not sufficient to revert this reduction (p = 1.5×10 −18 vs. control, p = 9.6×10 −29 vs. daf-2(1370) worms). Quantification of intestinal expression for each group in arbitrary units was achieved by measuring fluorescence levels in 20 images using ImageJ. All values indicate mean expression and error bars represent SEM. All p-values are t-test p-values. (C,D) egl-27 does not act upstream of daf-16 or elt-3 , as indicated by expression of a sod-3::GFP transcriptional reporter. (C) Representative images showing day 2 adult hermaphrodites grown at 20°C expressing sod-3::GFP . (D) Quantification of intestinal expression for each group in arbitrary units using imageJ to measure fluorescence from 15 images. sod-3::GFP expression is increased in daf-2 mutants compared to wild-type. Levels of sod-3::GFP expression both in control and daf-2(e1370) worms are unaffected by egl-27(we3) . (E,F) egl-27 regulates its own expression. (E) representative images showing egl-27::mCherry expression from day 2 adult hermaphrodites that were hatched at 20°C and grown at 15°C from day 1 of adulthood. (F) Quantification of intestinal expression for each group in arbitrary units using ImageJ to measure fluorescence intensity from 30 images. egl-27 expression is increased in egl-27(we3) mutants (p = 0.0015).
Figure Legend Snippet: egl-27 acts downstream of daf-2 /ILR, daf-16 /FOXO, and elt-3 /GATA. (A) representative images of egl-27::mCherry hermaphrodites at day 2 of adulthood showing median expression levels for each group. (B) egl-27 expression is increased in daf-2(e1370) mutants (p = 1.5×10 −11 vs. control) and this increase is suppressed in daf-2(e1370); daf-16(mu86) double mutants (p = 2.9×10 −17 vs. daf-2(e1370) worms). egl-27 expression is reduced in elt-3(vp1) mutants (p = 2.3×10 −8 vs. control) and the addition of daf-2(e1370) in daf-2(e1370); elt-3(vp1) double mutants is not sufficient to revert this reduction (p = 1.5×10 −18 vs. control, p = 9.6×10 −29 vs. daf-2(1370) worms). Quantification of intestinal expression for each group in arbitrary units was achieved by measuring fluorescence levels in 20 images using ImageJ. All values indicate mean expression and error bars represent SEM. All p-values are t-test p-values. (C,D) egl-27 does not act upstream of daf-16 or elt-3 , as indicated by expression of a sod-3::GFP transcriptional reporter. (C) Representative images showing day 2 adult hermaphrodites grown at 20°C expressing sod-3::GFP . (D) Quantification of intestinal expression for each group in arbitrary units using imageJ to measure fluorescence from 15 images. sod-3::GFP expression is increased in daf-2 mutants compared to wild-type. Levels of sod-3::GFP expression both in control and daf-2(e1370) worms are unaffected by egl-27(we3) . (E,F) egl-27 regulates its own expression. (E) representative images showing egl-27::mCherry expression from day 2 adult hermaphrodites that were hatched at 20°C and grown at 15°C from day 1 of adulthood. (F) Quantification of intestinal expression for each group in arbitrary units using ImageJ to measure fluorescence intensity from 30 images. egl-27 expression is increased in egl-27(we3) mutants (p = 0.0015).

Techniques Used: Expressing, Fluorescence, Activated Clotting Time Assay

egl-27 functions to promote longevity. (A) egl-27(we3) completely suppresses the longevity phenotype of daf-2(e1370) mutants. All worms were hatched at 20°C and shifted to 15°C at day 2 of adulthood. daf-2(e1370) : n = 279, mean lifespan = 48.9 days, median lifespan = 49 days, 95% mortality = 70 days. daf-2(e1370); daf-16(mu86) : n = 209, mean lifespan = 24.0 days, median lifespan = 25 days, 95% mortality = 30 days. daf-2(e1370); egl-27(we3) : n = 234, mean lifespan = 21.5 days, median lifespan = 20 days, 95% mortality = 36 days. p = 0; log rank test comparing daf-2(e1370); egl-27(we3) vs. daf-2(e1370). N2: n = 246, mean lifespan = 31.6 days, median lifespan = 31 days, 95% mortality = 41 days. egl-27(we3) : n = 232, mean lifespan = 27.8 days, median lifespan = 28 days, 95% mortality = 37 days. p = 7.8×10 −10 vs. N2. Additional lifespan data can be found in Table S1 . (B) OP177 and SD1601, two independent strains overexpressing egl-27 extend lifespan compared to SD1507, transgenic control worms. SD1507 (control): n = 165, mean lifespan = 26.3 days, median lifespan = 27 days, 95% mortality = 40 days. SD1601 ( egl-27::mCherry ): n = 80, mean lifespan = 31 days, median lifespan = 31 days, 95% mortality = 42 days. p-value = 4.0×10 −5 vs. control. OP177 ( egl-27::GFP ): n = 146, mean lifespan = 30.1 days, median lifespan = 30.5 days, 95% mortality = 44 days. p-value = 4.9×10 −5 vs. control. Additional lifespan data can be found in Table S1 . (C) egl::GFP rescues the larval arrest and embryonic lethality phenotypes of egl-27(we3) mutants. Error bars indicate SEM across 10 independent experiments. Total number of eggs across all 10 experiments for N2 = 167, egl-27(we3) = 154, egl-27(we3); egl-27::GFP = 164.
Figure Legend Snippet: egl-27 functions to promote longevity. (A) egl-27(we3) completely suppresses the longevity phenotype of daf-2(e1370) mutants. All worms were hatched at 20°C and shifted to 15°C at day 2 of adulthood. daf-2(e1370) : n = 279, mean lifespan = 48.9 days, median lifespan = 49 days, 95% mortality = 70 days. daf-2(e1370); daf-16(mu86) : n = 209, mean lifespan = 24.0 days, median lifespan = 25 days, 95% mortality = 30 days. daf-2(e1370); egl-27(we3) : n = 234, mean lifespan = 21.5 days, median lifespan = 20 days, 95% mortality = 36 days. p = 0; log rank test comparing daf-2(e1370); egl-27(we3) vs. daf-2(e1370). N2: n = 246, mean lifespan = 31.6 days, median lifespan = 31 days, 95% mortality = 41 days. egl-27(we3) : n = 232, mean lifespan = 27.8 days, median lifespan = 28 days, 95% mortality = 37 days. p = 7.8×10 −10 vs. N2. Additional lifespan data can be found in Table S1 . (B) OP177 and SD1601, two independent strains overexpressing egl-27 extend lifespan compared to SD1507, transgenic control worms. SD1507 (control): n = 165, mean lifespan = 26.3 days, median lifespan = 27 days, 95% mortality = 40 days. SD1601 ( egl-27::mCherry ): n = 80, mean lifespan = 31 days, median lifespan = 31 days, 95% mortality = 42 days. p-value = 4.0×10 −5 vs. control. OP177 ( egl-27::GFP ): n = 146, mean lifespan = 30.1 days, median lifespan = 30.5 days, 95% mortality = 44 days. p-value = 4.9×10 −5 vs. control. Additional lifespan data can be found in Table S1 . (C) egl::GFP rescues the larval arrest and embryonic lethality phenotypes of egl-27(we3) mutants. Error bars indicate SEM across 10 independent experiments. Total number of eggs across all 10 experiments for N2 = 167, egl-27(we3) = 154, egl-27(we3); egl-27::GFP = 164.

Techniques Used: Transgenic Assay

11) Product Images from "Kirrel3-mediated synapse formation is attenuated by disease-associated missense variants"

Article Title: Kirrel3-mediated synapse formation is attenuated by disease-associated missense variants

Journal: bioRxiv

doi: 10.1101/2019.12.30.891085

Kirrel3 expression does not increase axon-dendrite contact in cultured neurons. A, Representative images of neurons transfected with GFP and Kirrel3 (green) contacted by mCherry (mCh) labeled axons with (top) or without (bottom) Kirrel3. Scale bar = 10μm. B , Quantification of axon contact assay normalized to the Kirrel3-negative mCherry-labelled axon condition. Axon contact index is the area of mCherry positive axon per area GFP positive cell. n=22-29 neurons (indicated in each bar) from 2 cultures, p=0.2445 (Mann Whitney Test). ns = not significant.
Figure Legend Snippet: Kirrel3 expression does not increase axon-dendrite contact in cultured neurons. A, Representative images of neurons transfected with GFP and Kirrel3 (green) contacted by mCherry (mCh) labeled axons with (top) or without (bottom) Kirrel3. Scale bar = 10μm. B , Quantification of axon contact assay normalized to the Kirrel3-negative mCherry-labelled axon condition. Axon contact index is the area of mCherry positive axon per area GFP positive cell. n=22-29 neurons (indicated in each bar) from 2 cultures, p=0.2445 (Mann Whitney Test). ns = not significant.

Techniques Used: Expressing, Cell Culture, Transfection, Labeling, MANN-WHITNEY

Missense variants attenuate Kirrel3-mediated synapse formation. A , Representative images of CA1 dendrites transfected with GFP only (GFP), or GFP and indicated FLAG-Kirrel3 variants. Outlines show dendrite area as determined from GFP expression. DG pre-synapses (yellow) are identified by co-labelling of SPO (red) and VGluT1 (green). Arrows point to DG pre-synapses. Scale bar = 5μm. B , Quantification of DG pre-synapse density on CA1 dendrites normalized to wild-type Kirrel3 and with multiple comparisons to wild-type Kirrel3. n=30-55 neurons (indicated in each bar) from 3-5 cultures, (One-way ANOVA Kruskal-Wallis test with multiple comparisons to WT Kirrel3). C , Same data as ssssspresented in B but shown normalized to GFP and with multiple comparisons to GFP. See results section for specific p-values but *p
Figure Legend Snippet: Missense variants attenuate Kirrel3-mediated synapse formation. A , Representative images of CA1 dendrites transfected with GFP only (GFP), or GFP and indicated FLAG-Kirrel3 variants. Outlines show dendrite area as determined from GFP expression. DG pre-synapses (yellow) are identified by co-labelling of SPO (red) and VGluT1 (green). Arrows point to DG pre-synapses. Scale bar = 5μm. B , Quantification of DG pre-synapse density on CA1 dendrites normalized to wild-type Kirrel3 and with multiple comparisons to wild-type Kirrel3. n=30-55 neurons (indicated in each bar) from 3-5 cultures, (One-way ANOVA Kruskal-Wallis test with multiple comparisons to WT Kirrel3). C , Same data as ssssspresented in B but shown normalized to GFP and with multiple comparisons to GFP. See results section for specific p-values but *p

Techniques Used: Transfection, Expressing

Kirrel3 in a non-neuronal cell is not sufficient to mediate synapse formation. A , Representative images of the presynaptic neuron/HEK293 co-culture assay. Cultures were immunostained for GFP (blue) to label transfected HEK293 cells and VGluT1 (red) to label pre-synapses. Transfection conditions are indicated on each image. Nlgn1 = Neuroligin-1, K3 = Kirrel3. Arrow in bottom right panel points to an outline of a FLAG-Kirrel3 positive axon B , Quantification of presynaptic hemisynapse density from the co-culture assay normalized to the GFP-only negative control (GFP). n=32-61 cells (indicated in each bar) from 2 cultures, GFP vs Nlgn1 p=0.0074; GFP vs Kirrel3, p=0.3988; GFP vs K3+K3 p > 0.9999 (One-way ANOVA Kruskal-Wallis test, p=0.0226, with Dunn’s multiple comparisons). C, Images of the postsynaptic hemisynapse density from the co-culture assay. Cultures were immunostained with GFP (blue), vGluT1 (red) and PSD-95 (green). Postsynaptic hemisynapses are labelled by PSD-95 alone with no VGluT1. Nrxn1 = Neurexin-1. D , Quantification of postsynaptic hemisynapse density from co-culture assay normalized to the GFP-only negative control. n=19-20 cells (indicated in each bar) from one culture, GFP vs Nrxn1 p=0.0252; GFP vs Kirrel3 p > 0.9999 (One-way ANOVA Kruskal-Wallis test, p=0.0256, with Dunn’s multiple comparisons). Scale bar in A and C = 20μm.
Figure Legend Snippet: Kirrel3 in a non-neuronal cell is not sufficient to mediate synapse formation. A , Representative images of the presynaptic neuron/HEK293 co-culture assay. Cultures were immunostained for GFP (blue) to label transfected HEK293 cells and VGluT1 (red) to label pre-synapses. Transfection conditions are indicated on each image. Nlgn1 = Neuroligin-1, K3 = Kirrel3. Arrow in bottom right panel points to an outline of a FLAG-Kirrel3 positive axon B , Quantification of presynaptic hemisynapse density from the co-culture assay normalized to the GFP-only negative control (GFP). n=32-61 cells (indicated in each bar) from 2 cultures, GFP vs Nlgn1 p=0.0074; GFP vs Kirrel3, p=0.3988; GFP vs K3+K3 p > 0.9999 (One-way ANOVA Kruskal-Wallis test, p=0.0226, with Dunn’s multiple comparisons). C, Images of the postsynaptic hemisynapse density from the co-culture assay. Cultures were immunostained with GFP (blue), vGluT1 (red) and PSD-95 (green). Postsynaptic hemisynapses are labelled by PSD-95 alone with no VGluT1. Nrxn1 = Neurexin-1. D , Quantification of postsynaptic hemisynapse density from co-culture assay normalized to the GFP-only negative control. n=19-20 cells (indicated in each bar) from one culture, GFP vs Nrxn1 p=0.0252; GFP vs Kirrel3 p > 0.9999 (One-way ANOVA Kruskal-Wallis test, p=0.0256, with Dunn’s multiple comparisons). Scale bar in A and C = 20μm.

Techniques Used: Co-culture Assay, Transfection, Negative Control

Kirrel3 missense variants show normal surface localization. A , Schematic of Kirrel3 protein with approximate positions of disease-associated missense variants. Variants tested in this study are magenta. B , Representative images of CHO cells transfected with mCh-2A-FLAG-Kirrel3 variants. Cells were live-labelled with chicken-anti-FLAG (magenta) to show extracellular (EC) Kirrel3 protein and then permeabilized and stained with mouse-anti-FLAG (orange) to show intracellular (IC) Kirrel3 protein. Cell area was determined by GFP expression and is depicted by a white outline. Scale bar = 15μm. C , Quantification of EC/IC Kirrel3 protein levels normalized to wild-type Kirrel3. n=9 cells each from 3 cultures, p=0.4424 (One-way ANOVA). D , Representative images showing a dendrite from a mCherry-2A-FLAG-Kirrel3 wild-type transfected neuron. EC FLAG-Kirrel3 (yellow) localizes adjacent to juxtaposed synaptic markers VGluT1 (blue) and PSD-95 (red). mCherry-2A (magenta) serves as a cell marker. Scale bar = 4μm. E , Quantification of the percent of synapses (juxtaposed VGluT1 + , PSD-95 + puncta) with adjacent FLAG-Kirrel3 proteins for each variant. n=9-19 neurons (indicated in each bar) from two cultures, p=0.3197 (One-way ANOVA Kruskal-Wallis test). ns = not significant
Figure Legend Snippet: Kirrel3 missense variants show normal surface localization. A , Schematic of Kirrel3 protein with approximate positions of disease-associated missense variants. Variants tested in this study are magenta. B , Representative images of CHO cells transfected with mCh-2A-FLAG-Kirrel3 variants. Cells were live-labelled with chicken-anti-FLAG (magenta) to show extracellular (EC) Kirrel3 protein and then permeabilized and stained with mouse-anti-FLAG (orange) to show intracellular (IC) Kirrel3 protein. Cell area was determined by GFP expression and is depicted by a white outline. Scale bar = 15μm. C , Quantification of EC/IC Kirrel3 protein levels normalized to wild-type Kirrel3. n=9 cells each from 3 cultures, p=0.4424 (One-way ANOVA). D , Representative images showing a dendrite from a mCherry-2A-FLAG-Kirrel3 wild-type transfected neuron. EC FLAG-Kirrel3 (yellow) localizes adjacent to juxtaposed synaptic markers VGluT1 (blue) and PSD-95 (red). mCherry-2A (magenta) serves as a cell marker. Scale bar = 4μm. E , Quantification of the percent of synapses (juxtaposed VGluT1 + , PSD-95 + puncta) with adjacent FLAG-Kirrel3 proteins for each variant. n=9-19 neurons (indicated in each bar) from two cultures, p=0.3197 (One-way ANOVA Kruskal-Wallis test). ns = not significant

Techniques Used: Transfection, Staining, Expressing, Marker, Variant Assay

Ectopic Kirrel3 expression induces ectopic DG pre-synapses in cultured neurons. A , Schematic of Kirrel3 (K3) protein. Ig; Immunoglobulin domain. B , Schematic of the gain-of-function assay design. Top: In the control condition, a Kirrel3 positive DG neuron contacts a Kirrel3 negative CA1 dendrite and does not form a pre-synapse. Bottom: A Kirrel3 positive DG neuron contacts a Kirrel3-transfected CA1 neuron allowing for a DG pre-synapse to form. C , Tiled 20x image of an adult mouse hippocampal section immunostained with the CA1 marker Foxp1 (blue) and the presynaptic markers VGluT1 (green) and SPO (red). Grayscale images below. In each image the white arrow indicates approximate CA3 border. Scale bars = 200μm. D , Dendrites from cultured rat CA1 dendrites transfected with GFP (left) or GFP + Kirrel3 (right). GFP is not shown but is outlined in white. DG pre-synapses (yellow) are identified by co-labelling of SPO (red) and VGluT1 (green). Arrows point to DG pre-synapses. Scale bar = 5 μm. E , Quantification of DG pre-synapse density per length of CA1 dendrite and normalized to the GFP condition. n=38-48 neurons (indicated in each bar) from 3 cultures, p=0.0027 (Mann Whitney test). F , Quantification of CA synapse density per length of CA1 dendrite and normalized to the GFP condition. n=38-48 neurons (indicated in each bar) from 3 cultures, p=0.9552 (Mann Whitney test). G , Quantification of DG pre-synapse density per length of CA1 dendrite from Kirrel3 wild-type (WT) and knockout (KO) cultures. All are normalized to the wild-type GFP condition. n=17-42 neurons (indicated in each bar) from 3 cultures, WT: GFP vs GFP+Kirrel3 p=0.0088; KO: GFP vs GFP+Kirrel3, p=0.9560 (Two-way ANOVA with Sidak’s multiple comparisons, p= 0.0266; interaction) H , Schematic of WT versus KO assay results suggesting that Kirrel3 must be present in both the pre- and postsynaptic neuron to induce DG synapse formation. For F, and G, ns = not significant.
Figure Legend Snippet: Ectopic Kirrel3 expression induces ectopic DG pre-synapses in cultured neurons. A , Schematic of Kirrel3 (K3) protein. Ig; Immunoglobulin domain. B , Schematic of the gain-of-function assay design. Top: In the control condition, a Kirrel3 positive DG neuron contacts a Kirrel3 negative CA1 dendrite and does not form a pre-synapse. Bottom: A Kirrel3 positive DG neuron contacts a Kirrel3-transfected CA1 neuron allowing for a DG pre-synapse to form. C , Tiled 20x image of an adult mouse hippocampal section immunostained with the CA1 marker Foxp1 (blue) and the presynaptic markers VGluT1 (green) and SPO (red). Grayscale images below. In each image the white arrow indicates approximate CA3 border. Scale bars = 200μm. D , Dendrites from cultured rat CA1 dendrites transfected with GFP (left) or GFP + Kirrel3 (right). GFP is not shown but is outlined in white. DG pre-synapses (yellow) are identified by co-labelling of SPO (red) and VGluT1 (green). Arrows point to DG pre-synapses. Scale bar = 5 μm. E , Quantification of DG pre-synapse density per length of CA1 dendrite and normalized to the GFP condition. n=38-48 neurons (indicated in each bar) from 3 cultures, p=0.0027 (Mann Whitney test). F , Quantification of CA synapse density per length of CA1 dendrite and normalized to the GFP condition. n=38-48 neurons (indicated in each bar) from 3 cultures, p=0.9552 (Mann Whitney test). G , Quantification of DG pre-synapse density per length of CA1 dendrite from Kirrel3 wild-type (WT) and knockout (KO) cultures. All are normalized to the wild-type GFP condition. n=17-42 neurons (indicated in each bar) from 3 cultures, WT: GFP vs GFP+Kirrel3 p=0.0088; KO: GFP vs GFP+Kirrel3, p=0.9560 (Two-way ANOVA with Sidak’s multiple comparisons, p= 0.0266; interaction) H , Schematic of WT versus KO assay results suggesting that Kirrel3 must be present in both the pre- and postsynaptic neuron to induce DG synapse formation. For F, and G, ns = not significant.

Techniques Used: Expressing, Cell Culture, Functional Assay, Transfection, Marker, MANN-WHITNEY, Knock-Out

12) Product Images from "Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes"

Article Title: Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes

Journal: bioRxiv

doi: 10.1101/2020.07.20.212050

CSR-1a silences a repetitive somatic transgene. A) Schematic of the forward genetic screen used to uncover genes that repress rpn-2p::GFP expression. B) Viable alleles recovered from the forward genetic screen. C) Schematic representation of the csr-1a alleles recovered from the forward genetic screen. D) Fluorescence micrographs of the non-mutagenized reporter strain and the reporter after introduction of csr-1a [G120*] . Scale bar, 100μm. E) Box plot of normalized median relative fluorescence of the reporter strain and the reporter with csr-1a [G120*] strain (with Tukey whiskers). **** indicates significance of p
Figure Legend Snippet: CSR-1a silences a repetitive somatic transgene. A) Schematic of the forward genetic screen used to uncover genes that repress rpn-2p::GFP expression. B) Viable alleles recovered from the forward genetic screen. C) Schematic representation of the csr-1a alleles recovered from the forward genetic screen. D) Fluorescence micrographs of the non-mutagenized reporter strain and the reporter after introduction of csr-1a [G120*] . Scale bar, 100μm. E) Box plot of normalized median relative fluorescence of the reporter strain and the reporter with csr-1a [G120*] strain (with Tukey whiskers). **** indicates significance of p

Techniques Used: Expressing, Fluorescence

13) Product Images from "Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling"

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling

Journal: bioRxiv

doi: 10.1101/826735

Spatiotemporal coordination of hgfa and met expression controls vagus motor axon target selection, see also Supplementary Figure 2 . (A-C) Double hgfa (purple) and tcf21 (brown) RNA in situ hybridization time series showing A-P sequential expression in the PAs. Arrowheads indicate hgfa expression in PAs. Numbers mark PAs. (D-F) met RNA in situ hybridization time series showing A-P expansion over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (G) Quantification of met expression domains over time represented in (D-F). Data represent mean ± SEM. t-test ****P
Figure Legend Snippet: Spatiotemporal coordination of hgfa and met expression controls vagus motor axon target selection, see also Supplementary Figure 2 . (A-C) Double hgfa (purple) and tcf21 (brown) RNA in situ hybridization time series showing A-P sequential expression in the PAs. Arrowheads indicate hgfa expression in PAs. Numbers mark PAs. (D-F) met RNA in situ hybridization time series showing A-P expansion over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (G) Quantification of met expression domains over time represented in (D-F). Data represent mean ± SEM. t-test ****P

Techniques Used: Expressing, Selection, RNA In Situ Hybridization

(A-C) hoxb5a RNA in situ hybridization time series showing hoxb5a expression receding towards the posterior over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (D) Quantification of hoxb5a expression domains over time represented in (A-C). Data represent mean ± SEM. t-test ****P
Figure Legend Snippet: (A-C) hoxb5a RNA in situ hybridization time series showing hoxb5a expression receding towards the posterior over time in the mX nucleus. mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. (D) Quantification of hoxb5a expression domains over time represented in (A-C). Data represent mean ± SEM. t-test ****P

Techniques Used: RNA In Situ Hybridization, Expressing

Retinoic Acid is a putative regulator of A-P vagus motor neuron identity (A-B) Differential gene expression between anterior and posterior mX neurons. (A) Representative anterior (A) and posterior (A’) photoconverted (magenta) regions collected for RNAseq analysis. (B) Volcano plot of RNAseq data indicating mRNAs enriched in anterior (left) or posterior (right) mX neurons. Dashed lines indicate significance threshold for a false discovery rate of 10% (y-axis) and a fold change of 1.5 (x-axis). Blue and red dots represent significantly differentially expressed genes. Red dots represent genes indicative of differential Retinoic Acid signaling between populations. (C-E) RNA In situ hybridization of selected genes identified in (B). In each image, mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. hoxb5a (C) and dhrs3b (D) mRNAs are enriched in posterior mX neurons. crabp1b (E) mRNA is enriched in anterior mX neurons. (F) The retinoic acid-responsive RARE:GFP transgene (green) is expressed in posterior, but not anterior, mX neurons (magenta). All images are lateral views oriented with anterior to left. Scale bars = 50 μm.
Figure Legend Snippet: Retinoic Acid is a putative regulator of A-P vagus motor neuron identity (A-B) Differential gene expression between anterior and posterior mX neurons. (A) Representative anterior (A) and posterior (A’) photoconverted (magenta) regions collected for RNAseq analysis. (B) Volcano plot of RNAseq data indicating mRNAs enriched in anterior (left) or posterior (right) mX neurons. Dashed lines indicate significance threshold for a false discovery rate of 10% (y-axis) and a fold change of 1.5 (x-axis). Blue and red dots represent significantly differentially expressed genes. Red dots represent genes indicative of differential Retinoic Acid signaling between populations. (C-E) RNA In situ hybridization of selected genes identified in (B). In each image, mRNA expression is purple and the mX neurons marked by isl1:GFP are brown. The A-P span of the mX nucleus is indicated by the curved dotted line. hoxb5a (C) and dhrs3b (D) mRNAs are enriched in posterior mX neurons. crabp1b (E) mRNA is enriched in anterior mX neurons. (F) The retinoic acid-responsive RARE:GFP transgene (green) is expressed in posterior, but not anterior, mX neurons (magenta). All images are lateral views oriented with anterior to left. Scale bars = 50 μm.

Techniques Used: Expressing, RNA In Situ Hybridization

14) Product Images from "Novel replication complex architecture in rubella replicon‐transfected cells"

Article Title: Novel replication complex architecture in rubella replicon‐transfected cells

Journal: Cellular Microbiology

doi: 10.1111/j.1462-5822.2006.00837.x

Cellular distribution of replicase components, C, lysosomes and actin in replicon‐transfected cells (replicon‐transfected BHK‐21 cells maintained for several weeks after drug selection). Single optical sections are shown. Cells were transfected with RUBrep/C‐GFP/neo in A–D and F and with RUBrep/GFP/neo in E. An asterisk marks the centre of the cell nucleus in the micrographs. A, B and D. Antibody labelling of P150 (A and D) or P90 (B), both in red, with C‐GFP (green), with colocalization shown in white (A and C) or yellow (D) and indicated by arrows. C. Arrows indicate C‐GFP (green) surrounding lysosomes (red, visualized with Lysotracker). Filamentous patterns of colocalization are denoted in D. E and F. Colocalization (indicated by arrows) of actin (labelled with phalloidin‐blue) and P150 (E, antibody labelled in red) or C‐GFP (F, green).
Figure Legend Snippet: Cellular distribution of replicase components, C, lysosomes and actin in replicon‐transfected cells (replicon‐transfected BHK‐21 cells maintained for several weeks after drug selection). Single optical sections are shown. Cells were transfected with RUBrep/C‐GFP/neo in A–D and F and with RUBrep/GFP/neo in E. An asterisk marks the centre of the cell nucleus in the micrographs. A, B and D. Antibody labelling of P150 (A and D) or P90 (B), both in red, with C‐GFP (green), with colocalization shown in white (A and C) or yellow (D) and indicated by arrows. C. Arrows indicate C‐GFP (green) surrounding lysosomes (red, visualized with Lysotracker). Filamentous patterns of colocalization are denoted in D. E and F. Colocalization (indicated by arrows) of actin (labelled with phalloidin‐blue) and P150 (E, antibody labelled in red) or C‐GFP (F, green).

Techniques Used: Transfection, Selection

Organelle distribution and ultrastructure in replicon‐transfected cells (control non‐transfected BHK‐21 cells, BHK‐21 cells stably transfected with RUBrep/C‐GFP/neo, and Vero cells transiently transfected with this replicon at 2 days post transfection). Single optical sections are shown. Vero cells are shown in A–C while the rest of fields are of BHK‐21 cells. The cells were stained for lysosomes (blue) (A–C), Golgi (red) and mitochondria (visualized with Mitotracker blue) (D–F) and RER (red) (G–J). Panels B/C, E/F and I/J show the same field; in C, F and J, the green signal has been removed to allow better resolution of the organelle staining pattern. (K) EM of perinuclear areas of cells transfected with RUBrep/C‐GFP/neo. Dense, modified lysosome‐like organelles or CPVs (‘RC’ for replication complex) locally recruit mitochondria (mi) in the proximity of the cell nucleus (N) and are surrounded by RER, and Golgi stacks (G). Bar: 1 μm.
Figure Legend Snippet: Organelle distribution and ultrastructure in replicon‐transfected cells (control non‐transfected BHK‐21 cells, BHK‐21 cells stably transfected with RUBrep/C‐GFP/neo, and Vero cells transiently transfected with this replicon at 2 days post transfection). Single optical sections are shown. Vero cells are shown in A–C while the rest of fields are of BHK‐21 cells. The cells were stained for lysosomes (blue) (A–C), Golgi (red) and mitochondria (visualized with Mitotracker blue) (D–F) and RER (red) (G–J). Panels B/C, E/F and I/J show the same field; in C, F and J, the green signal has been removed to allow better resolution of the organelle staining pattern. (K) EM of perinuclear areas of cells transfected with RUBrep/C‐GFP/neo. Dense, modified lysosome‐like organelles or CPVs (‘RC’ for replication complex) locally recruit mitochondria (mi) in the proximity of the cell nucleus (N) and are surrounded by RER, and Golgi stacks (G). Bar: 1 μm.

Techniques Used: Transfection, Stable Transfection, Staining, Modification

Association of RUB replicase components with CPVs by immuno‐EM in BHK cells transfected with RUBrep/C‐GFP/neo after drug selection (A–D) and confocal microscopy detection of dsRNA after saponin or SLO permeabilization in BHK cells transfected with RUBrep/GFP/neo after drug selection (E–G). Immunogold labelling with anti‐P150 (A–C) and anti‐P90 (D) antibodies demonstrate the presence of these replicase components in CPVs at the EM level. Labelling of vacuoles within the CPVs is denoted by arrowheads, the straight elements in side views by stars, and labelling of straight elements by arrows. (E–F) Confocal microscopy localization of dsRNA (red) after total permeabilization with saponin (E) or plasma membrane permeabilization with SLO (F and G). Z‐series projections of single optical sections are shown. The cell on the right side in F, which was not permeabilized with SLO treatment, maintained the cytosolic GFP and did not show dsRNA labelling. Under these conditions only the molecules exposed to the cytoplasmic side are detected. White asterisks mark cell nuclei. Bars: 200 nm.
Figure Legend Snippet: Association of RUB replicase components with CPVs by immuno‐EM in BHK cells transfected with RUBrep/C‐GFP/neo after drug selection (A–D) and confocal microscopy detection of dsRNA after saponin or SLO permeabilization in BHK cells transfected with RUBrep/GFP/neo after drug selection (E–G). Immunogold labelling with anti‐P150 (A–C) and anti‐P90 (D) antibodies demonstrate the presence of these replicase components in CPVs at the EM level. Labelling of vacuoles within the CPVs is denoted by arrowheads, the straight elements in side views by stars, and labelling of straight elements by arrows. (E–F) Confocal microscopy localization of dsRNA (red) after total permeabilization with saponin (E) or plasma membrane permeabilization with SLO (F and G). Z‐series projections of single optical sections are shown. The cell on the right side in F, which was not permeabilized with SLO treatment, maintained the cytosolic GFP and did not show dsRNA labelling. Under these conditions only the molecules exposed to the cytoplasmic side are detected. White asterisks mark cell nuclei. Bars: 200 nm.

Techniques Used: Transfection, Selection, Confocal Microscopy

Ultrastructural analysis of CPVs in replicon‐transfected and RUB‐infected cells by conventional processing for thin‐section and EM (BHK‐21 cells transfected with RUBrep/C‐GFP/neo after drug selection). A and B. Conventional EM shows two views of electron‐dense CPVs in perinuclear areas of replicon‐transfected cells. Internal vesicles (V) and straight elements (arrows) are denoted as are locally recruited organelles, RER and mitochondria (mi). The CPVs in B appear to be side views of the CPVs while in A, a frontal view of the CPV is shown. C and D. CPVs in RUB‐infected BHK cells 24 (C) and 48 (D) hours post infection. C is a higher magnification while D is a lower magnification. CPVs in infected cells contain both vesicles and straight elements (arrows). Inset in D shows a characteristic dense lysosome in a control cell. V, vesicles; N, nucleus. Bars: 200 nm in A–C; 0.5 μm in D.
Figure Legend Snippet: Ultrastructural analysis of CPVs in replicon‐transfected and RUB‐infected cells by conventional processing for thin‐section and EM (BHK‐21 cells transfected with RUBrep/C‐GFP/neo after drug selection). A and B. Conventional EM shows two views of electron‐dense CPVs in perinuclear areas of replicon‐transfected cells. Internal vesicles (V) and straight elements (arrows) are denoted as are locally recruited organelles, RER and mitochondria (mi). The CPVs in B appear to be side views of the CPVs while in A, a frontal view of the CPV is shown. C and D. CPVs in RUB‐infected BHK cells 24 (C) and 48 (D) hours post infection. C is a higher magnification while D is a lower magnification. CPVs in infected cells contain both vesicles and straight elements (arrows). Inset in D shows a characteristic dense lysosome in a control cell. V, vesicles; N, nucleus. Bars: 200 nm in A–C; 0.5 μm in D.

Techniques Used: Transfection, Infection, Selection

Association of dsRNA, C and mitochondrial protein P32 with CPVs in replicon‐transfected cells by immuno‐EM (BHK cells transfected with RUBrep/C‐GFP/neo after drug selection). A. Immunogold labelling with anti‐dsRNA antibodies. dsRNA signal associated with a straight element (star) is indicated with an arrow. B–D. Immunogold labelling with anti‐GFP antibodies to detect C‐GFP. Labelling around vacuoles (asterisks) and straight elements (stars) within CPVs is indicated by arrows. In C, exterior labelling of a mitochondrion (mi) is indicated by an arrowhead. E. Immunogold labelling with anti‐P32 antibodies. Labelling of the interior of a CPV is shown with arrows in both the main field and inset (straight elements are indicated by a star). Note the labelling of a neighbouring mitochondrion (mi). Bars: 200 nm.
Figure Legend Snippet: Association of dsRNA, C and mitochondrial protein P32 with CPVs in replicon‐transfected cells by immuno‐EM (BHK cells transfected with RUBrep/C‐GFP/neo after drug selection). A. Immunogold labelling with anti‐dsRNA antibodies. dsRNA signal associated with a straight element (star) is indicated with an arrow. B–D. Immunogold labelling with anti‐GFP antibodies to detect C‐GFP. Labelling around vacuoles (asterisks) and straight elements (stars) within CPVs is indicated by arrows. In C, exterior labelling of a mitochondrion (mi) is indicated by an arrowhead. E. Immunogold labelling with anti‐P32 antibodies. Labelling of the interior of a CPV is shown with arrows in both the main field and inset (straight elements are indicated by a star). Note the labelling of a neighbouring mitochondrion (mi). Bars: 200 nm.

Techniques Used: Transfection, Selection

Immunofluorescence and confocal microscopy localization of RUB replication components in replicon‐transfected cells (Vero cells, 2 or 4 days post transfection). Single optical sections are shown. An asterisk marks the centre of the cell nucleus. A. Schematic diagrams of the two replicons used in this study, RUBrep/GFP/neo and RUBrep/C‐GFP/neo. ORFs are represented by boxes and untranslated regions by lines. B. Localization of P150 non‐structural protein (red) and lysosomes (blue) in cells transfected with RUBrep/GFP/neo. Lysosomes were labelled with anti‐LAMP‐1 antibodies. Arrows indicate P150 signal within lysosomes. With this construct, GFP localizes in both the cell nucleus (*) and the cytoplasm. C. Localization of dsRNA (antibody labelled in red) and lysosomes (blue) in cells transfected with RUBrep/GFP/neo. Arrows indicate dsRNA signal within lysosomes. D and E. Localization of dsRNA (red) and lysosomes (blue) in cells transfected with RUBrep/C‐GFP/neo. In D, arrows indicate dsRNA signal within lysosomes while in E, arrows indicate localization of dsRNA signal on cell periphery. With this construct, the C‐GFP fusion accumulates in the cytoplasm. F and G. Two confocal single sections of the same cell transfected with RUBrep/C‐GFP/neo and stained for dsRNA (red) showing dsRNA concentrating in the perinuclear region in the central section (F) or on the cell periphery in the lower section (G).
Figure Legend Snippet: Immunofluorescence and confocal microscopy localization of RUB replication components in replicon‐transfected cells (Vero cells, 2 or 4 days post transfection). Single optical sections are shown. An asterisk marks the centre of the cell nucleus. A. Schematic diagrams of the two replicons used in this study, RUBrep/GFP/neo and RUBrep/C‐GFP/neo. ORFs are represented by boxes and untranslated regions by lines. B. Localization of P150 non‐structural protein (red) and lysosomes (blue) in cells transfected with RUBrep/GFP/neo. Lysosomes were labelled with anti‐LAMP‐1 antibodies. Arrows indicate P150 signal within lysosomes. With this construct, GFP localizes in both the cell nucleus (*) and the cytoplasm. C. Localization of dsRNA (antibody labelled in red) and lysosomes (blue) in cells transfected with RUBrep/GFP/neo. Arrows indicate dsRNA signal within lysosomes. D and E. Localization of dsRNA (red) and lysosomes (blue) in cells transfected with RUBrep/C‐GFP/neo. In D, arrows indicate dsRNA signal within lysosomes while in E, arrows indicate localization of dsRNA signal on cell periphery. With this construct, the C‐GFP fusion accumulates in the cytoplasm. F and G. Two confocal single sections of the same cell transfected with RUBrep/C‐GFP/neo and stained for dsRNA (red) showing dsRNA concentrating in the perinuclear region in the central section (F) or on the cell periphery in the lower section (G).

Techniques Used: Immunofluorescence, Confocal Microscopy, Transfection, Construct, Staining

Ultrastructural analysis of modified lysosomes in replicon‐transfected cells prepared by freeze substitution (BHK cells transfected with RUBrep/C‐GFP/neo after drug selection). A. Large CPV with numerous filled vesicles (arrows) and straight elements (stars). B. Higher magnification of a CPV with small, filled vesicles that appear to contain an internal core (arrow). C. Small vesicular elements are abundant inside the dense content of some CPVs, both on the periphery (arrows) and internal areas. D and E. High magnification of straight elements showing dense, rigid membranes (arrows in D, arrowhead in E). White arrow in E points to a stack of rigid membranes. F and G. Sections along the straight sheets (rigid membranes) show features compatible with a close packing of particles (arrows). V: vesicles. Bars: 200 nm in A; 50 nm in B, D and E; 100 nm in C, F and G.
Figure Legend Snippet: Ultrastructural analysis of modified lysosomes in replicon‐transfected cells prepared by freeze substitution (BHK cells transfected with RUBrep/C‐GFP/neo after drug selection). A. Large CPV with numerous filled vesicles (arrows) and straight elements (stars). B. Higher magnification of a CPV with small, filled vesicles that appear to contain an internal core (arrow). C. Small vesicular elements are abundant inside the dense content of some CPVs, both on the periphery (arrows) and internal areas. D and E. High magnification of straight elements showing dense, rigid membranes (arrows in D, arrowhead in E). White arrow in E points to a stack of rigid membranes. F and G. Sections along the straight sheets (rigid membranes) show features compatible with a close packing of particles (arrows). V: vesicles. Bars: 200 nm in A; 50 nm in B, D and E; 100 nm in C, F and G.

Techniques Used: Modification, Transfection, Selection

15) Product Images from "Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis"

Article Title: Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20215334

Schematic representation of the plasmid pMDC85/ cry10Aa . The 12,838 bp-long plasmid consists of the  cry10Aa  gene under 2XCaMV35S (cauliflower mosaic virus 35S promoter with a duplicated enhancer region) and 3′NOS (nopaline synthase terminator); HygR (hygromycin phosphotransferase) gene ( hph ) under 2XCaMV35S and 3′NOS terminator, mGFP (modified green fluorescent protein) under 2XCaMV35S promoter and 3′NOS terminator. 6xHis (6x histidine affinity tag); attB1-B2 (mutant version of  att B); RB (right border repeat from nopaline C58T-DNA); pVS1 StaA (stability protein from  Pseudomonas  plasmid pVS1); pVS1 RepA (replication protein from  Pseudomonas  plasmid pVS1); Bom (bases of mobility region from pBR322); Ori (high-copy-number ColE1/pMB1/pBR322/pUC origin of replication); KanR (aminoglycoside phosphotransferase  aphA-3 , which confers kanamycin resistance); LB T-DNA repeat (left border repeat from nopaline C58 T-DNA); CaMV poly(A) signal (cauliflower mosaic virus polyadenylation signal); CAP binding site ( E. coli  catabolite activator protein); lac promoter (three segments) promoter for the  E. coli lac  operon; lac operator (laccase repressor encoded by  lacI ); and M13 rev. (common sequencing primer).
Figure Legend Snippet: Schematic representation of the plasmid pMDC85/ cry10Aa . The 12,838 bp-long plasmid consists of the cry10Aa gene under 2XCaMV35S (cauliflower mosaic virus 35S promoter with a duplicated enhancer region) and 3′NOS (nopaline synthase terminator); HygR (hygromycin phosphotransferase) gene ( hph ) under 2XCaMV35S and 3′NOS terminator, mGFP (modified green fluorescent protein) under 2XCaMV35S promoter and 3′NOS terminator. 6xHis (6x histidine affinity tag); attB1-B2 (mutant version of att B); RB (right border repeat from nopaline C58T-DNA); pVS1 StaA (stability protein from Pseudomonas plasmid pVS1); pVS1 RepA (replication protein from Pseudomonas plasmid pVS1); Bom (bases of mobility region from pBR322); Ori (high-copy-number ColE1/pMB1/pBR322/pUC origin of replication); KanR (aminoglycoside phosphotransferase aphA-3 , which confers kanamycin resistance); LB T-DNA repeat (left border repeat from nopaline C58 T-DNA); CaMV poly(A) signal (cauliflower mosaic virus polyadenylation signal); CAP binding site ( E. coli catabolite activator protein); lac promoter (three segments) promoter for the E. coli lac operon; lac operator (laccase repressor encoded by lacI ); and M13 rev. (common sequencing primer).

Techniques Used: Plasmid Preparation, Modification, Mutagenesis, Binding Assay, Sequencing

( A ) C. arabica embryogenic calli induced from leaf explants used for stable genetic transformation. ( B ) Hygromycin-resistant embryogenic calli of C. arabica derived from bombarded globular structures after four months of selection, showing independent events of genetic transformation. ( C ) Somatic embryos under osmotic stress in the first month of maturation. ( D , E ) Individual somatic embryo under osmotic stress in the second month of maturation. Scale bar 1000 μm. ( F ) Green fluorescent protein (GFP) stable expression in nuclei of transgenic mature somatic embryos of C. arabica , after three months of growth on 50 mg/L hygromycin-containing medium. ( G ) Control somatic embryo, showing no GFP expression. ( H ) Regenerated plants of C. arabica after six months in soil conditions. Wild-type plants in red pots, transgenic plants in green and blue pots.
Figure Legend Snippet: ( A ) C. arabica embryogenic calli induced from leaf explants used for stable genetic transformation. ( B ) Hygromycin-resistant embryogenic calli of C. arabica derived from bombarded globular structures after four months of selection, showing independent events of genetic transformation. ( C ) Somatic embryos under osmotic stress in the first month of maturation. ( D , E ) Individual somatic embryo under osmotic stress in the second month of maturation. Scale bar 1000 μm. ( F ) Green fluorescent protein (GFP) stable expression in nuclei of transgenic mature somatic embryos of C. arabica , after three months of growth on 50 mg/L hygromycin-containing medium. ( G ) Control somatic embryo, showing no GFP expression. ( H ) Regenerated plants of C. arabica after six months in soil conditions. Wild-type plants in red pots, transgenic plants in green and blue pots.

Techniques Used: Transformation Assay, Derivative Assay, Selection, Expressing, Transgenic Assay

16) Product Images from "The SCFDia2 Ubiquitin E3 Ligase Ubiquitylates Sir4 and Functions in Transcriptional Silencing"

Article Title: The SCFDia2 Ubiquitin E3 Ligase Ubiquitylates Sir4 and Functions in Transcriptional Silencing

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002846

Dia2 functions in parallel with Rtt106 in HMR silencing. (A–C) Mutant dia2 Δ cells have defects in transcriptional silencing at the HMR locus. Cells with the indicated genotype were analyzed for GFP expression using flow cytometry (A), and the percentage of cells expressing GFP was reported (B). Wild-type (WT) cells (less than 1%) and cells with SIR3 deleted ( sir3 Δ, over 95%) were used as standards to set the gate for GFP expression. The flow cytometry profile and percentage of cells expressing GFP are reported from one of three independent experiments. (C) HMR a1 gene expression is elevated in dia2 Δ and dia2Δ rtt106 Δ mutants. RNA was collected from cells of the indicated genotype and reverse transcribed. Expression of the HMR a1 gene was analyzed via real-time PCR, normalized against the expression of the ACT1 gene and reported as the average ± standard deviation (s.d.) of two independent experiments. (D–E) Telomere silencing is compromised in dia2 Δ cells. (D) Telomere silencing was assayed using the URA3 gene integrated at the left end telomere of chromosome VII and growth on medium containing 5-fluoroorotic acid (FOA). (E) YFR057W expression levels were determined by real time RT-PCR and analyzed as described above with the average ± s.d. of two independent experiments shown.
Figure Legend Snippet: Dia2 functions in parallel with Rtt106 in HMR silencing. (A–C) Mutant dia2 Δ cells have defects in transcriptional silencing at the HMR locus. Cells with the indicated genotype were analyzed for GFP expression using flow cytometry (A), and the percentage of cells expressing GFP was reported (B). Wild-type (WT) cells (less than 1%) and cells with SIR3 deleted ( sir3 Δ, over 95%) were used as standards to set the gate for GFP expression. The flow cytometry profile and percentage of cells expressing GFP are reported from one of three independent experiments. (C) HMR a1 gene expression is elevated in dia2 Δ and dia2Δ rtt106 Δ mutants. RNA was collected from cells of the indicated genotype and reverse transcribed. Expression of the HMR a1 gene was analyzed via real-time PCR, normalized against the expression of the ACT1 gene and reported as the average ± standard deviation (s.d.) of two independent experiments. (D–E) Telomere silencing is compromised in dia2 Δ cells. (D) Telomere silencing was assayed using the URA3 gene integrated at the left end telomere of chromosome VII and growth on medium containing 5-fluoroorotic acid (FOA). (E) YFR057W expression levels were determined by real time RT-PCR and analyzed as described above with the average ± s.d. of two independent experiments shown.

Techniques Used: Mutagenesis, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation, Quantitative RT-PCR

17) Product Images from "Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins"

Article Title: Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.571807

Intracellular distribution and structure formation from ectopic expression of the dichorhavirus MPs, Ns, and Ps. The CiLV-N and OFV-citrus MP, N, and P fused at their C-termini with eGFP ( ) were co-expressed with mRFP( ) nucleus marker or aniline blue ( ) fluorochrome (plasmodesmata marker) in epidermal cells of N. benthamiana . Fluorescence signals were captured 48–72 h post-infiltration with confocal microscope Zeiss LSM 780 model. The green (GFP), red (mRFP) channels, and merged images are shown for each protein expressed with nucleus marker. The blue (aniline blue), green, transmitted light (T.L), and merged images are shown for MP expressed with plasmodesma marker. The dotted line in the transmitted light image indicates the cell wall (CW). Cyt = cytoplasm. The numbers at the top of cell panels correspond to the number of fluorescent cells showing fluorescence in the nucleus, i.e., 25/100 indicates that 25 out of 100 fluorescent cells showed the presence of GFP in the nuclei. The free-eGFP expression is located as a diffuse signal distributed in the cytoplasm and nucleus (Ba) . (A) Image shows the CiLV-N MP- expression in punctate structures into the nuclei co-localizing with nuclei marker (a) , and a signal distributed at cell membrane periphery, colocalized in punctate structures with plasmodesmata along the cell periphery (c) . (b) OFV-citrus MP- expression in a diffuse signal evenly distributed into the nucleus co-localizing with nuclei marker, and co-localizing in punctate structures with plasmodesmata along the cell periphery (d) . (B) CiLV-N and OFV-citrus N- expression in a diffuse signal distributed within some cell nuclei co-localizing with nuclei marker ( b and d ). GFP-empty nuclei are also visualized ( c and e ). N- signal is also distributed throughout the cytoplasm for both viruses. (C) The P- expression is visualized exclusively in cell nuclei colocalized with nuclei marker for both CiLV-N (a) and OFV-citrus (b) . Images below show in higher magnification the respective nuclei indicated in the red dashed boxes. Red and white bars correspond to 10 and 50 μm, respectively.
Figure Legend Snippet: Intracellular distribution and structure formation from ectopic expression of the dichorhavirus MPs, Ns, and Ps. The CiLV-N and OFV-citrus MP, N, and P fused at their C-termini with eGFP ( ) were co-expressed with mRFP( ) nucleus marker or aniline blue ( ) fluorochrome (plasmodesmata marker) in epidermal cells of N. benthamiana . Fluorescence signals were captured 48–72 h post-infiltration with confocal microscope Zeiss LSM 780 model. The green (GFP), red (mRFP) channels, and merged images are shown for each protein expressed with nucleus marker. The blue (aniline blue), green, transmitted light (T.L), and merged images are shown for MP expressed with plasmodesma marker. The dotted line in the transmitted light image indicates the cell wall (CW). Cyt = cytoplasm. The numbers at the top of cell panels correspond to the number of fluorescent cells showing fluorescence in the nucleus, i.e., 25/100 indicates that 25 out of 100 fluorescent cells showed the presence of GFP in the nuclei. The free-eGFP expression is located as a diffuse signal distributed in the cytoplasm and nucleus (Ba) . (A) Image shows the CiLV-N MP- expression in punctate structures into the nuclei co-localizing with nuclei marker (a) , and a signal distributed at cell membrane periphery, colocalized in punctate structures with plasmodesmata along the cell periphery (c) . (b) OFV-citrus MP- expression in a diffuse signal evenly distributed into the nucleus co-localizing with nuclei marker, and co-localizing in punctate structures with plasmodesmata along the cell periphery (d) . (B) CiLV-N and OFV-citrus N- expression in a diffuse signal distributed within some cell nuclei co-localizing with nuclei marker ( b and d ). GFP-empty nuclei are also visualized ( c and e ). N- signal is also distributed throughout the cytoplasm for both viruses. (C) The P- expression is visualized exclusively in cell nuclei colocalized with nuclei marker for both CiLV-N (a) and OFV-citrus (b) . Images below show in higher magnification the respective nuclei indicated in the red dashed boxes. Red and white bars correspond to 10 and 50 μm, respectively.

Techniques Used: Expressing, Marker, Fluorescence, Microscopy

Related Articles

Clone Assay:

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Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Selection:

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Fluorescence:

Article Title: Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages ▿
Article Snippet: .. To quantify the percentages of BMDMs carrying live bacteria at different times postinfection, after GFP expression was induced for 1 h, sequential images of green fluorescence and phase contrast were taken from a random field by use of a Zeiss Axiovert S100 microscope with a 32× objective. ..

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Microscopy:

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling
Article Snippet: .. Sparse transgenic labeling of cells was done by injecting one-cell-stage embryos with 50pg of plasmid and 50pg of Tol2 mRNA, and screening embryos for sparse GFP expression at 2 days post fertilization on a Zeiss AxioZoom.V16 microscope. .. Generation of met and hgfa mutant alleles met and hgfa mutant alleles were generated using CRISPR/Cas9 as described in ( ) using the short oligo method with the following modifications: gRNAs were designed using chopchop ( https://chopchop.cbu.uib.no ).

Article Title: Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages ▿
Article Snippet: .. To quantify the percentages of BMDMs carrying live bacteria at different times postinfection, after GFP expression was induced for 1 h, sequential images of green fluorescence and phase contrast were taken from a random field by use of a Zeiss Axiovert S100 microscope with a 32× objective. ..

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Transgenic Assay:

Article Title: Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper
Article Snippet: .. For the resolution of GFP expression from transgenic pepper, a confocal image analyzer program (Carl Zeiss LSM Browser Bersion 2.80.1123; Jena, Germany) was used to detect the green color which appeared only under GFP expression. .. PCR and RT-PCR To detect the transgene in transformed pepper plants by PCR, the total DNA of transformed pepper was isolated using a DNA extraction kit (iNtRon Biotechnology, http://www.intronbio.com ).

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling
Article Snippet: .. Sparse transgenic labeling of cells was done by injecting one-cell-stage embryos with 50pg of plasmid and 50pg of Tol2 mRNA, and screening embryos for sparse GFP expression at 2 days post fertilization on a Zeiss AxioZoom.V16 microscope. .. Generation of met and hgfa mutant alleles met and hgfa mutant alleles were generated using CRISPR/Cas9 as described in ( ) using the short oligo method with the following modifications: gRNAs were designed using chopchop ( https://chopchop.cbu.uib.no ).

Confocal Laser Scanning Microscopy:

Article Title: Sugar administration is an effective adjunctive therapy in the treatment of Pseudomonas aeruginosa pneumonia
Article Snippet: .. GFP-expressing or LIVE/DEAD stained PAO1 and PDO300 were examined by LCSM (LSM 510; Carl Zeiss MicroImaging) at a magnification of ×600. ..

Labeling:

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling
Article Snippet: .. Sparse transgenic labeling of cells was done by injecting one-cell-stage embryos with 50pg of plasmid and 50pg of Tol2 mRNA, and screening embryos for sparse GFP expression at 2 days post fertilization on a Zeiss AxioZoom.V16 microscope. .. Generation of met and hgfa mutant alleles met and hgfa mutant alleles were generated using CRISPR/Cas9 as described in ( ) using the short oligo method with the following modifications: gRNAs were designed using chopchop ( https://chopchop.cbu.uib.no ).

Expressing:

Article Title: Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper
Article Snippet: .. For the resolution of GFP expression from transgenic pepper, a confocal image analyzer program (Carl Zeiss LSM Browser Bersion 2.80.1123; Jena, Germany) was used to detect the green color which appeared only under GFP expression. .. PCR and RT-PCR To detect the transgene in transformed pepper plants by PCR, the total DNA of transformed pepper was isolated using a DNA extraction kit (iNtRon Biotechnology, http://www.intronbio.com ).

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling
Article Snippet: .. Sparse transgenic labeling of cells was done by injecting one-cell-stage embryos with 50pg of plasmid and 50pg of Tol2 mRNA, and screening embryos for sparse GFP expression at 2 days post fertilization on a Zeiss AxioZoom.V16 microscope. .. Generation of met and hgfa mutant alleles met and hgfa mutant alleles were generated using CRISPR/Cas9 as described in ( ) using the short oligo method with the following modifications: gRNAs were designed using chopchop ( https://chopchop.cbu.uib.no ).

Article Title: Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages ▿
Article Snippet: .. To quantify the percentages of BMDMs carrying live bacteria at different times postinfection, after GFP expression was induced for 1 h, sequential images of green fluorescence and phase contrast were taken from a random field by use of a Zeiss Axiovert S100 microscope with a 32× objective. ..

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Article Title: Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing
Article Snippet: .. After hatching, adult males were shifted to 29°C for three days to allow GFP expression. ssxΔ , w+ , Sxl-T2A-GAL4(3xP3-RFP) or Sxl-T2A-GAL4(3xP3-RFP) males expressing either GFP or Stinger were cold anesthetized, screened for GFP expression with a Leica M2 Fl III and imaged with a Zeiss Axiophot combined with a Zeiss Colibri. .. Sister-of-Sex-lethal is expressed in both sexes In Drosophila melanogaster, Sxl mRNA isoforms are expressed in a sex-specific fashion resulting in the production of functional, full-length protein exclusively in female animals.

Article Title: Combinatorial Action of Temporally Segregated Transcription Factors
Article Snippet: .. The following day, L1 animals were mounted on a glass slide covered with a 5% agarose pad and examined for GFP expression in ASEs using a Zeiss Axio Imager.Z2 with sCMOS camera, Sola SM2 solid state white light excitation system and 40x/1.3 EC plan-neofluar Oil DIC objective. .. RNA Interference (RNAi) RNA interference assays were performed by feeding worms with bacteria expressing dsRNA as described in ( ).

Staining:

Article Title: Sugar administration is an effective adjunctive therapy in the treatment of Pseudomonas aeruginosa pneumonia
Article Snippet: .. GFP-expressing or LIVE/DEAD stained PAO1 and PDO300 were examined by LCSM (LSM 510; Carl Zeiss MicroImaging) at a magnification of ×600. ..

Western Blot:

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Immunofluorescence:

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection
Article Snippet: .. Puromycin-resistant clones were pooled to avoid confounds introduced by clonal selection, and p21 or GFP expression was verified by Western blot analysis and GFP immunofluorescence under an inverted fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany). .. Primary mixed cortical neurons were infected with adenovirus (multiplicity of infection = 100) harboring both p21 and GFP cDNAs (Adp21+GFP), or GFP cDNA (Ad-GFP) alone.

Plasmid Preparation:

Article Title: Retinoic acid organizes the vagus motor topographic map via spatiotemporal regulation of Hgf/Met signaling
Article Snippet: .. Sparse transgenic labeling of cells was done by injecting one-cell-stage embryos with 50pg of plasmid and 50pg of Tol2 mRNA, and screening embryos for sparse GFP expression at 2 days post fertilization on a Zeiss AxioZoom.V16 microscope. .. Generation of met and hgfa mutant alleles met and hgfa mutant alleles were generated using CRISPR/Cas9 as described in ( ) using the short oligo method with the following modifications: gRNAs were designed using chopchop ( https://chopchop.cbu.uib.no ).

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    Carl Zeiss gfp expressing cells gfp expressing animals
    <t>Promoter-GFP</t> reporter genes confirm neural expression of transcripts from pan-neural enriched data sets . Transgenic animals expressing GFP reporters for representative transcripts exclusively enriched in either the IVT derived data set ( A-D ) or the WT-Pico-amplified sample ( E-L ). A . F32B6.11 ::GFP is expressed throughout the C. elegans nervous system including neurons associated with the Nerve ring in the head, motor neurons throughout the Ventral Nerve Cord (VNC) and in tail ganglia. D . F49H12.4 ::GFP is selectively expressed in PVD nociceptive neuron and in two additional neurons in the tail region. Note the highly branched PVD dendritic architecture (arrowheads). E . Differential Interference Contrast (DIC) image of midbody region of 2 nd stage larva ( F.) expressing F47B8.3 ::GFP in GABAergic motor neurons (DD5, VD10, VD11) in the ventral nerve cord. P9 and P10 denote landmark hypodermal blast cells. G-H . ZC155.2 ::GFP and C50F4.4 ::GFP are expressed in VNC motor neurons (e.g. VA10, VB11, etc). Anterior to left, Ventral down. VNC (Ventral Nerve Cord).
    Gfp Expressing Cells Gfp Expressing Animals, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Zeiss neonatal rat cardiomyocytes expressing α actinin 2 gfp variants
    Mutations Affecting Regulation of <t>α-Actinin-2</t> with PIP2 Do Not Influence F-Actin Binding but Impact α-Actinin-2 Z-Disk Dynamics (A) Binding of α-actinin-2 variants to F-actin and titin Zr-7. α-actinin-2 WT, NEECK, and PIP2 mutants (PIP2 mut) were cosedimented with actin, and equal amounts of supernatant (s) and pellet (p) fractions were subjected to SDS-PAGE and visualized by Coomassie blue. (B and C) FRAP measurements of α-actinin-2 dynamics in live NRCs expressing <t>GFP-labeled</t> α-actinin-2 variants (WT, PIP2 mutants, and NEECK). (B) Snapshots at prebleach and two time points postbleach; the bleached region of interest (ROI) is highlighted by a dotted box. Note that NEECK fluorescence does not recover within the 144 s time course shown here, whereas rapid recovery is observed for WT α-actinin. Insets: ROIs enlarged 2-fold. (C) Quantification of fluorescence intensity recovery. Note that the slowed fluorescence recovery of the PIP2 mutant is mirrored by treatment with 500 μM neomycin (Neo). Bold lines, exponential fits; shaded lines, average values. Error bars indicate SD.
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    Promoter-GFP reporter genes confirm neural expression of transcripts from pan-neural enriched data sets . Transgenic animals expressing GFP reporters for representative transcripts exclusively enriched in either the IVT derived data set ( A-D ) or the WT-Pico-amplified sample ( E-L ). A . F32B6.11 ::GFP is expressed throughout the C. elegans nervous system including neurons associated with the Nerve ring in the head, motor neurons throughout the Ventral Nerve Cord (VNC) and in tail ganglia. D . F49H12.4 ::GFP is selectively expressed in PVD nociceptive neuron and in two additional neurons in the tail region. Note the highly branched PVD dendritic architecture (arrowheads). E . Differential Interference Contrast (DIC) image of midbody region of 2 nd stage larva ( F.) expressing F47B8.3 ::GFP in GABAergic motor neurons (DD5, VD10, VD11) in the ventral nerve cord. P9 and P10 denote landmark hypodermal blast cells. G-H . ZC155.2 ::GFP and C50F4.4 ::GFP are expressed in VNC motor neurons (e.g. VA10, VB11, etc). Anterior to left, Ventral down. VNC (Ventral Nerve Cord).

    Journal: BMC Genomics

    Article Title: Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system

    doi: 10.1186/1471-2164-9-84

    Figure Lengend Snippet: Promoter-GFP reporter genes confirm neural expression of transcripts from pan-neural enriched data sets . Transgenic animals expressing GFP reporters for representative transcripts exclusively enriched in either the IVT derived data set ( A-D ) or the WT-Pico-amplified sample ( E-L ). A . F32B6.11 ::GFP is expressed throughout the C. elegans nervous system including neurons associated with the Nerve ring in the head, motor neurons throughout the Ventral Nerve Cord (VNC) and in tail ganglia. D . F49H12.4 ::GFP is selectively expressed in PVD nociceptive neuron and in two additional neurons in the tail region. Note the highly branched PVD dendritic architecture (arrowheads). E . Differential Interference Contrast (DIC) image of midbody region of 2 nd stage larva ( F.) expressing F47B8.3 ::GFP in GABAergic motor neurons (DD5, VD10, VD11) in the ventral nerve cord. P9 and P10 denote landmark hypodermal blast cells. G-H . ZC155.2 ::GFP and C50F4.4 ::GFP are expressed in VNC motor neurons (e.g. VA10, VB11, etc). Anterior to left, Ventral down. VNC (Ventral Nerve Cord).

    Article Snippet: Microscopy and identification of GFP expressing cells GFP-expressing animals were visualized by Differential Interference Contrast (DIC) and epifluorescence optics in either a Zeiss Axioplan or Axiovert compound microscope.

    Techniques: Expressing, Transgenic Assay, Derivative Assay, Amplification

    Overexpression of GDP-locked Rab41 partially inhibited VSV-G transport from ER to Golgi apparatus. Wild type HeLa cells were microinjected with either 25 ng/µl plasmid encoding the tsO45 mutant of VSV-G-GFP (Control) or a mixture of 100 ng/µl myc-tagged GDP-locked Rab41 encoding plasmid and 25 ng/µl tsO45 mutant of VSV-G-GFP encoding plasmid. After 24-h incubation at 39.5° C, VSV-G was accumulated in the ER (A, upper panel). Cells were then shifted to 32° C, permissive conditions for VSV-G transport, and incubated for 40 min in the presence of cycloheximide to prevent further protein synthesis (A, lower panel). Cells were then fixed and visualized by wide field light microscopy. At the end of a 40-min chase, Golgi accumulation of VSV-G was observed in both control and GDP-locked Rab41 overexpressing cells. However, VSV-G retention in the ER for GDP-locked Rab41 overexpressing cells was much higher than that in control cells (A, lower panel, and B). Successful co-injection was confirmed by antibody staining. All images shown or used for quantification were single-plane deconvolved. Error bars represent the mean ± SEM of ~20 injected cells. Asterisks mark injected cells.

    Journal: PLoS ONE

    Article Title: Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

    doi: 10.1371/journal.pone.0071886

    Figure Lengend Snippet: Overexpression of GDP-locked Rab41 partially inhibited VSV-G transport from ER to Golgi apparatus. Wild type HeLa cells were microinjected with either 25 ng/µl plasmid encoding the tsO45 mutant of VSV-G-GFP (Control) or a mixture of 100 ng/µl myc-tagged GDP-locked Rab41 encoding plasmid and 25 ng/µl tsO45 mutant of VSV-G-GFP encoding plasmid. After 24-h incubation at 39.5° C, VSV-G was accumulated in the ER (A, upper panel). Cells were then shifted to 32° C, permissive conditions for VSV-G transport, and incubated for 40 min in the presence of cycloheximide to prevent further protein synthesis (A, lower panel). Cells were then fixed and visualized by wide field light microscopy. At the end of a 40-min chase, Golgi accumulation of VSV-G was observed in both control and GDP-locked Rab41 overexpressing cells. However, VSV-G retention in the ER for GDP-locked Rab41 overexpressing cells was much higher than that in control cells (A, lower panel, and B). Successful co-injection was confirmed by antibody staining. All images shown or used for quantification were single-plane deconvolved. Error bars represent the mean ± SEM of ~20 injected cells. Asterisks mark injected cells.

    Article Snippet: For quantification of VSV-G distribution, cells expressing VSV-G-GFP were wide-field imaged using a 63x/1.40 numerical aperture objective and a Zeiss 200M inverted microscope.

    Techniques: Over Expression, Plasmid Preparation, Mutagenesis, Incubation, Light Microscopy, Injection, Staining

    Depletion of Rab41 partially inhibited VSV-G transport from ER to the cell surface with a significant inhibition of ER to Golgi transport. Wild type HeLa cells were incubated with either siRab41(4) or non-targeting siRNA duplexes for 96 h and then transfected with plasmid encoding VSV-G-GFP. At the end of the 39.5° C incubation period, VSV-G was accumulated in the ER (A). Cells were then shifted to 32° C, permissive conditions for VSV-G transport, and incubated for various chase time in the presence of cycloheximide to prevent further protein synthesis (B). Cells were then fixed and cell surface stained for VSV-G, and visualized by wide field light microscopy. At the end of a 20-min chase or 40-min chase, Golgi accumulation of VSV-G was observed in both control and Rab41 knockdown cells. However, VSV-G retention in the ER of Rab41 depleted cells was decidedly higher than that of control cells (total VSV-G, left two columns in B, and C). Consistently, at later chase times, surface accumulation of VSV-G in Rab41 knockdown cells was quantitatively slower than that in control cells (surface VSV-G, right two columns in B, and D). All images shown or used for quantification were single-plane deconvolved. Error bars are the mean ± St Dev of three independent experiments. ~30 cells were assayed for each time point in the individual experiments.

    Journal: PLoS ONE

    Article Title: Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

    doi: 10.1371/journal.pone.0071886

    Figure Lengend Snippet: Depletion of Rab41 partially inhibited VSV-G transport from ER to the cell surface with a significant inhibition of ER to Golgi transport. Wild type HeLa cells were incubated with either siRab41(4) or non-targeting siRNA duplexes for 96 h and then transfected with plasmid encoding VSV-G-GFP. At the end of the 39.5° C incubation period, VSV-G was accumulated in the ER (A). Cells were then shifted to 32° C, permissive conditions for VSV-G transport, and incubated for various chase time in the presence of cycloheximide to prevent further protein synthesis (B). Cells were then fixed and cell surface stained for VSV-G, and visualized by wide field light microscopy. At the end of a 20-min chase or 40-min chase, Golgi accumulation of VSV-G was observed in both control and Rab41 knockdown cells. However, VSV-G retention in the ER of Rab41 depleted cells was decidedly higher than that of control cells (total VSV-G, left two columns in B, and C). Consistently, at later chase times, surface accumulation of VSV-G in Rab41 knockdown cells was quantitatively slower than that in control cells (surface VSV-G, right two columns in B, and D). All images shown or used for quantification were single-plane deconvolved. Error bars are the mean ± St Dev of three independent experiments. ~30 cells were assayed for each time point in the individual experiments.

    Article Snippet: For quantification of VSV-G distribution, cells expressing VSV-G-GFP were wide-field imaged using a 63x/1.40 numerical aperture objective and a Zeiss 200M inverted microscope.

    Techniques: Inhibition, Incubation, Transfection, Plasmid Preparation, Staining, Light Microscopy

    DAXX K/D increases autophagic flux in cultured PCa cells PC3. A , PC3 cells, control (WT) and DAXX K/D (K/D), transiently transfected with GFP-LC3 construct for 24 h, were cultured under normal ( Basal ) conditions as described under “Experimental

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Repressor DAXX Promotes Prostate Cancer Tumorigenicity via Suppression of Autophagy *

    doi: 10.1074/jbc.M115.658765

    Figure Lengend Snippet: DAXX K/D increases autophagic flux in cultured PCa cells PC3. A , PC3 cells, control (WT) and DAXX K/D (K/D), transiently transfected with GFP-LC3 construct for 24 h, were cultured under normal ( Basal ) conditions as described under “Experimental

    Article Snippet: Z -Stack confocal images of GFP-LC3-expressing cells were collected using the ×63 oil objective on a Zeiss LSM 710 microscope (Waitt Advanced Biophotonics Center, Salk Institute).

    Techniques: Cell Culture, Transfection, Construct

    Mutations Affecting Regulation of α-Actinin-2 with PIP2 Do Not Influence F-Actin Binding but Impact α-Actinin-2 Z-Disk Dynamics (A) Binding of α-actinin-2 variants to F-actin and titin Zr-7. α-actinin-2 WT, NEECK, and PIP2 mutants (PIP2 mut) were cosedimented with actin, and equal amounts of supernatant (s) and pellet (p) fractions were subjected to SDS-PAGE and visualized by Coomassie blue. (B and C) FRAP measurements of α-actinin-2 dynamics in live NRCs expressing GFP-labeled α-actinin-2 variants (WT, PIP2 mutants, and NEECK). (B) Snapshots at prebleach and two time points postbleach; the bleached region of interest (ROI) is highlighted by a dotted box. Note that NEECK fluorescence does not recover within the 144 s time course shown here, whereas rapid recovery is observed for WT α-actinin. Insets: ROIs enlarged 2-fold. (C) Quantification of fluorescence intensity recovery. Note that the slowed fluorescence recovery of the PIP2 mutant is mirrored by treatment with 500 μM neomycin (Neo). Bold lines, exponential fits; shaded lines, average values. Error bars indicate SD.

    Journal: Cell

    Article Title: The Structure and Regulation of Human Muscle α-Actinin

    doi: 10.1016/j.cell.2014.10.056

    Figure Lengend Snippet: Mutations Affecting Regulation of α-Actinin-2 with PIP2 Do Not Influence F-Actin Binding but Impact α-Actinin-2 Z-Disk Dynamics (A) Binding of α-actinin-2 variants to F-actin and titin Zr-7. α-actinin-2 WT, NEECK, and PIP2 mutants (PIP2 mut) were cosedimented with actin, and equal amounts of supernatant (s) and pellet (p) fractions were subjected to SDS-PAGE and visualized by Coomassie blue. (B and C) FRAP measurements of α-actinin-2 dynamics in live NRCs expressing GFP-labeled α-actinin-2 variants (WT, PIP2 mutants, and NEECK). (B) Snapshots at prebleach and two time points postbleach; the bleached region of interest (ROI) is highlighted by a dotted box. Note that NEECK fluorescence does not recover within the 144 s time course shown here, whereas rapid recovery is observed for WT α-actinin. Insets: ROIs enlarged 2-fold. (C) Quantification of fluorescence intensity recovery. Note that the slowed fluorescence recovery of the PIP2 mutant is mirrored by treatment with 500 μM neomycin (Neo). Bold lines, exponential fits; shaded lines, average values. Error bars indicate SD.

    Article Snippet: Fluorescence Recovery after Photobleaching Neonatal rat cardiomyocytes expressing α-actinin-2-GFP variants were imaged 30 h after transfection with a 63 × objective and analyzed with the FRAP module of a Zeiss LSM510 Meta microscope.

    Techniques: Binding Assay, SDS Page, Expressing, Labeling, Fluorescence, Mutagenesis

    PIP2 and Vinculin in Relation to the Z-Disk and Temporal Increase of the Titin T12 Epitope in α-Actinin WT- and NEECK-Transfected Cardiomyocytes, Related to Figures 5 and 6 (A) PIP2 is localized to the cardiac Z-disk. Immunofluorescence in untransfected NRC using PIP2 antibody shows a striated pattern that coincides with the Z-disk titin epitope T12 (arrows). Note the noncardiomyocytes above the striated cell, in which PIP2 is localized in a vesicular pattern. (B) The α-actinin-2 NEECK mutant does not colocalize in abnormal Z-disks. GFP-labeled NEECK α-actinin was transiently expressed in NRC for 18-48 hr. The NEECK mutant leads to widening of the Z-disk (upper row) and ultimately formation of rod-like structures (lower row). Note that vinculin shows no appreciable colocalization with α-actinin-2 but remains predominantly restricted to focal adhesions. Red: monoclonal anti-vinculin stain, green: α-actinin-2 NEECK-GFP, Blue: actin (Alexa643-phalloidine). (C) The optically resolvable T12 distance was measured in confocal immunofluorescence images 2 and 3 days after transfection of the GFP-labeled constructs. Note the expansion to an average width of over 600 nm at day 2 and over 900 nm at day 3, while the optical T12 distance in WT α-actinin transfected cells does not exceed 300 nm. Data from 9 independent cells each. Dashed lines: means.

    Journal: Cell

    Article Title: The Structure and Regulation of Human Muscle α-Actinin

    doi: 10.1016/j.cell.2014.10.056

    Figure Lengend Snippet: PIP2 and Vinculin in Relation to the Z-Disk and Temporal Increase of the Titin T12 Epitope in α-Actinin WT- and NEECK-Transfected Cardiomyocytes, Related to Figures 5 and 6 (A) PIP2 is localized to the cardiac Z-disk. Immunofluorescence in untransfected NRC using PIP2 antibody shows a striated pattern that coincides with the Z-disk titin epitope T12 (arrows). Note the noncardiomyocytes above the striated cell, in which PIP2 is localized in a vesicular pattern. (B) The α-actinin-2 NEECK mutant does not colocalize in abnormal Z-disks. GFP-labeled NEECK α-actinin was transiently expressed in NRC for 18-48 hr. The NEECK mutant leads to widening of the Z-disk (upper row) and ultimately formation of rod-like structures (lower row). Note that vinculin shows no appreciable colocalization with α-actinin-2 but remains predominantly restricted to focal adhesions. Red: monoclonal anti-vinculin stain, green: α-actinin-2 NEECK-GFP, Blue: actin (Alexa643-phalloidine). (C) The optically resolvable T12 distance was measured in confocal immunofluorescence images 2 and 3 days after transfection of the GFP-labeled constructs. Note the expansion to an average width of over 600 nm at day 2 and over 900 nm at day 3, while the optical T12 distance in WT α-actinin transfected cells does not exceed 300 nm. Data from 9 independent cells each. Dashed lines: means.

    Article Snippet: Fluorescence Recovery after Photobleaching Neonatal rat cardiomyocytes expressing α-actinin-2-GFP variants were imaged 30 h after transfection with a 63 × objective and analyzed with the FRAP module of a Zeiss LSM510 Meta microscope.

    Techniques: Transfection, Immunofluorescence, Mutagenesis, Labeling, Staining, Construct

    Constitutively Activated α-Actinin-2 Disrupts Z-Disks and Leads to Myofibril Disassembly GFP-labeled WT and NEECK α-actinin-2 were transiently expressed in NRCs for 18–48 hr. (A) WT α-actinin shows normal Z-disk localization, and the titin T12 epitope is resolved as a single line in standard confocal microscopy. (B) In contrast, NEECK leads to widening of the Z-disk and splitting of the T12 epitope after ∼18 hr (asterisk); doublet T12 lines are highlighted by arrows. (C) After 48 hr, Z-disks are completely disrupted and Z-disk titin, actin, and mutant α-actinin are localized in rod-like structures. (D and E) Superresolution microscopy reveals that epitopes of N-terminal Z1Z2 of titin and their ligand telethonin are unresolvable in WT-transfected cells; NEECK causes widening of Z-disks. Doublet Z1Z2/telethonin lines are highlighted by arrows and the central α-actinin region is indicated by arrowheads. Insets show 2-fold enlargement. Z-disk titin (T12 epitope) or telethonin, red; mutant α-actinin-GFP, green; actin (Alexa 688-phalloidin) or titin Z1Z2, blue.

    Journal: Cell

    Article Title: The Structure and Regulation of Human Muscle α-Actinin

    doi: 10.1016/j.cell.2014.10.056

    Figure Lengend Snippet: Constitutively Activated α-Actinin-2 Disrupts Z-Disks and Leads to Myofibril Disassembly GFP-labeled WT and NEECK α-actinin-2 were transiently expressed in NRCs for 18–48 hr. (A) WT α-actinin shows normal Z-disk localization, and the titin T12 epitope is resolved as a single line in standard confocal microscopy. (B) In contrast, NEECK leads to widening of the Z-disk and splitting of the T12 epitope after ∼18 hr (asterisk); doublet T12 lines are highlighted by arrows. (C) After 48 hr, Z-disks are completely disrupted and Z-disk titin, actin, and mutant α-actinin are localized in rod-like structures. (D and E) Superresolution microscopy reveals that epitopes of N-terminal Z1Z2 of titin and their ligand telethonin are unresolvable in WT-transfected cells; NEECK causes widening of Z-disks. Doublet Z1Z2/telethonin lines are highlighted by arrows and the central α-actinin region is indicated by arrowheads. Insets show 2-fold enlargement. Z-disk titin (T12 epitope) or telethonin, red; mutant α-actinin-GFP, green; actin (Alexa 688-phalloidin) or titin Z1Z2, blue.

    Article Snippet: Fluorescence Recovery after Photobleaching Neonatal rat cardiomyocytes expressing α-actinin-2-GFP variants were imaged 30 h after transfection with a 63 × objective and analyzed with the FRAP module of a Zeiss LSM510 Meta microscope.

    Techniques: Labeling, Confocal Microscopy, Mutagenesis, Microscopy, Transfection